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Fluorescent magnetic nanoprobes: Design and application for cell imaging
Journal of Colloid and Interface Science 351 (2010) 128–133
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Fluorescent magnetic nanoprobes: Design and application for cell imaging Guo Zhang a,c, Jianghua Feng b,**, Lehui Lu a,*, Baohua Zhang a,c, Linyuan Cao a,c a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China State Key Laboratory of Magnetic Resonance and Molecular and Atomic Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, Hubei 430071, PR China c Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China b
a r t i c l e
i n f o
Article history: Received 31 May 2010 Accepted 21 July 2010 Available online 27 July 2010 Keywords: Nanoprobes Magnetic Fluorescent T2-relaxivity Cell imaging
a b s t r a c t Multifunctional nanoprobes combining magnetic nanoparticles with organic dyes have attracted tremendous interest due to their promising applications in biomedical field. Here we demonstrate a facile and general strategy for the fabrication of robust fluorescent magnetic nanoprobes with high payloads of dye molecules and their use as multimodal nanoprobes for cell imaging. These nanoprobes not only effectively keep photochemical stability of dyes, but also provide a platform for grafting other functional or targeted moieties into silica surface via primary amines. Moreover, the nanoprobes are uniformly spherical morphology and can be dispersed well in aqueous solution, which are very desirable for biomedical applications. Importantly, this method can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Magnetic resonance imaging (MRI) is one of the most powerful and non-invasive imaging techniques by measuring proton relaxation processes of water in biological systems. In order to meet the different demands of biomedical research, Fe3O4 nanoparticles [1– 6], as MRI contrast enhancement agents [7–10], have been employed as a core platform to construct multimodal probes by the conjugation with other functional moieties including optical tags, radionuclides, targeting moiety, and drugs or gene carriers [11– 15]. Among these nanoprobes, Fe3O4 nanoparticles-based MRIfluorescent nanoprobes are of growing interest [16–19]. These MRI-fluorescent nanoprobes not only bridge gaps in sensitivity and depth of imaging between the two modalities, but also simultaneously obtain multiple imaging information data. Fluorescent molecules such as organic dyes could be directly conjugated to magnetic nanoparticles to produce MRI-fluorescent nanoprobes. These nanoprobes work nicely, but have the potential adversities including photobleaching and rapid fluorescence quenching effects [20]. To improve the stability and versatility of the fluorescent magnetic nanoprobes, desirable surface coating materials (e.g. polymer, dextran, etc.) or functional molecules with specific targeting groups (e.g. antibodies, peptides, etc.) are required. Also, silica has already served as candidate to encapsulate nanoprobes
* Corresponding author. Fax: +86 431 85262406. ** Corresponding author. E-mail addresses: [email protected] (J. Feng), [email protected] (L. Lu). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.07.056
because of its stability, biocompatibility, easy functionalization, and low cytotoxicity [21–27]. Silica-coated fluorescent magnetic nanoprobes have been recently reported by many research groups [28–35]. These diverse nanoprobes allow the development of biomedical platforms for simultaneous imaging, diagnosis and therapy. However, it is challenging to fabricate fluorescent magnetic nanoprobes with high photostability, high payloads of dyes and desirable outer surface for further modifications with functional or target moieties. To address the above issues, we put forward a design strategy of fluorescent magnetic nanoprobes. This strategy is facile and general for the synthesis of multifunctional nanoprobes combining magnetic Fe3O4 nanoparticles and dyes with isothiocyanate group. The as-synthesized nanoprobes including Fe3O4(FITC)@SiO2–NH2 and Fe3O4(RBITC)@SiO2–NH2 possess excellent fluorescent and magnetic properties, and good water solubility. Investigation on T2-relaxivity and cell fluorescence imaging of Fe3O4(RBITC)@SiO2–NH2 nanoprobes indicates that these nanoprobes are desirable labeling materials and may find potential applications in biomedical field. 2. Material and methods 2.1. Materials All chemicals were of analytic grade and used without further purification unless indicated. 1-Octadecene (ODE, 90%), oleic acid (90%), APTES, RBITC, polyoxyethylene (5) nonylphenyl ether (Igepal CO-520), ammonia solution (28%) and TEOS were
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purchased from Aldrich. FITC was purchased from Fluka. The water used was purified through a Millipore system. 2.2. Synthesis of Fe3O4 nanoparticles Monodisperse 12-nm Fe3O4 nanoparticles were synthesized by high-temperature thermal decomposition iron–oleate complex [36]. Firstly, iron–oleate complex was prepared by a reaction of ferric chloride (FeCl36H2O, 1.08 g, 4 mmol) and sodium oleate (3.65 g, 12 mmol) in a mixture solvent composed ethanol (8 mL), water (6 mL) and hexane (14 mL) at 70 °C for 4 h. Next, the upper organic layer containing iron–oleate complex was washed three times with water (10 mL) in a separatory funnel after completing the reaction, resulting in iron–oleate complex in a waxy solid form. The as-synthesized iron–oleate complex (3.6 g, 4 mmol) and oleic acid (0.57 g, 2 mmol) were dissolved in ODE (20 g) at room temperature and were heated to 320 °C with a heating rate of 3.3 °C min 1, and then kept for 30 min. After cooling to room temperature, the resulting Fe3O4 nanoparticles were precipitated by adding ethanol (50 mL), separated by centrifugation, and finally dried by vacuum at 80 °C. 2.3. Synthesis of FITC–APTES (or RBITC–APTES) conjugates FITC (5 mg) was conjugated with APTES (10 lL) in anhydrous ethanol (3 mL) by an addition reaction of isothiocyanate group with amine group. The reaction was carried out in dark for 12 h by slowly stirring under argon atmosphere. The reaction solution of FITC–APTES conjugates was then stored at 4 °C. Under identical conditions, RBITC–APTES conjugates were synthesized by an addition reaction of RBITC (13.8 mg) with APTES (10 lL) in anhydrous ethanol (3 mL) and stored at 4 °C. 2.4. Synthesis of Fe3O4(FITC)@SiO2–NH2 nanoprobes Fe3O4(FITC)@SiO2–NH2 nanoprobes were prepared by an improved microemulsion method [37]. Briefly, Igepal CO-520 (0.46 mL, 1 mmol) was dispersed into cyclohexane (9 mL) in a 50-mL three-necked flask by ultrasonication for 20 min. Under mechanical stirring, Fe3O4 cyclohexane solution (1 mg mL 1, 1 mL) and FITC–APTES conjugates (20 lL) were added into the microemulsion. Then, ammonia solution (80 lL) and TEOS (60 lL) was added, the mixture was allowed to age for 24 h to hydrolysis and condensation of the silica precursor. The final surface layer incorporating primary amines was formed by adding APTES (0.15 lL) and stirring for 24 h. The resulting Fe3O4(FITC)@SiO2–NH2 nanoprobes were collected by centrifugation and finally washed with ethanol three times and water three times, respectively. Similarly, using RBITC–APTES conjugates (20 lL) instead of FITC–APTES conjugates, Fe3O4(RBITC)@SiO2–NH2 can be made. 2.5. The measurement of T2-relaxivity (R2) Fe3O4(FITC)@SiO2–NH2 nanoparticles were dispersed in deuterated water (D2O). Five samples were prepared separately with the iron concentration of 0, 0.05, 0.1, 0.25, and 0.5 mM. Their transverse relaxation time (T2) was measured by using Carr–Purcell– Meiboom–Gill (CPMG) pulse sequence at 11.7 T and 25 °C using an NMR spectrometer. The relaxivities (R2) were obtained from the linear regressions (the relaxation rates (1/T2) vs. iron concentration). 2.6. Cell-uptake of Fe3O4(FITC)@SiO2–NH2 nanoprobes HeLa cells were seeded at a density of 6.0 104 cells/3 mL in a 6-well plate containing coverslips. Cell culture was maintained at
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37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) medium for 24 h. To determine cell-uptake of Fe3O4(FITC)@SiO2– NH2 nanoprobes, the cells were incubated with 10 lg mL 1 (the concentration of Fe) Fe3O4(FITC)@SiO2–NH2 nanoprobes in phosphate buffered saline (PBS) dispersion solution at 37 °C for 3 h. After washing with warm of PBS solution (2 mL), the cell sample was soaked into 4% PFA (para-formaldehyde) for 15 min. Following this, sample slips were washed with PBS and H2O several times, then dried. Finally, coverslips were mounted on drop of neutral balsam, leaving to dry at room temperature in the dark for confocal microscopy. 2.7. Characterization TEM images were obtained on an H-8100 TEM operating at 200 kV. XRD of the powder samples were examined on a D8 Focus diffractometer (Bruker) using Cu Ka radiation (k = 0.15405 nm). Magnetic studies were carried out on a Quantum Design SQUID magnetometer. Fluorescence photographs were taken on a Canon digital camera. The excitation and emission spectra were collected on an F-4500 spectrophotometer equipped with a 150 W xenon lamp as the excitation source. CLSM images were obtained on a CLSM (Leica TCS SP2). T2-relaxivity was measured at 11.7 T using an NMR spectrometer (Bruker AVII-500). 3. Results and discussion The synthetic process of the fluorescent magnetic Fe3O4(FITC)@SiO2–NH2 nanoprobes was shown in Scheme 1. Typically, the as-synthesized Fe3O4 dispersion and fluorescein isothiocyanate–3-aminopropyltriethoxysilane (FITC–APTES) conjugates were simultaneously added into a microemulsion, and APTES was added into the above microemulsion after 24 h. FITC–APTES conjugates were prepared in advance via an addition reaction between the isothiocyanate group of FITC dye and the primary amines group of APTES. This synthetic process enables the co-encapsulation of Fe3O4 nanoparticles and large amount of FITC dye molecules together via silica shell (Fig. S1a), and the grafting of primary amines into the silica surface. Such a core–shell structure has some advantages. First, it can prevent fluorescence quenching of the nanoprobes because FITC dye is not directly conjugated to Fe3O4 nanoparticles via chemical reaction (Fig. S1b) [38]; second, it can keep photochemical stability and increase fluorescence intensity of the dyes due to ‘‘caging effects” of silica shell [39–43]; third, it bears the potential to facilitate surface modification via primary amines that are more active than silanol groups. The amine site is an important surface-bound moiety to graft specific functional groups and biomolecules for biomedicine applications. The as-synthesized nanoprobes can be well-dispersed in aqueous solution, thus make them feasible to measure relaxivity and run the cell experiments. Transmission electron microscopy (TEM) images of Fe3O4 nanoparticles and Fe3O4(FITC)@SiO2–NH2 nanoprobes were shown in Fig. 1. Fe3O4 nanoparticles prepared via high-temperature thermal decomposition method were monodisperse with an average diameter of 12 nm (Fig. 1a). A thick layer of silica shell was then formed in situ through hydrolysis of tetraethyl orthosilicate (TEOS) and subsequent condensation of silica onto the surface of Fe3O4 cores, which was determined to be 8 nm. The thickness of the silica shell can be tuned by varying the volume of TEOS. A thick silica shell was preferred in this case since a thicker silica shell allowed high payloads of dye molecules to be achieved (Fig. S2). The final surface layer incorporating primary amines was formed in situ through hydrolysis of APTES. TEM image of as-synthesized Fe3O4(FITC)@SiO2–NH2 nanoprobes showed well-defined spherical core–shell
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Scheme 1. Schematic representation of the synthetic process, structure and property of Fe3O4(FITC)@SiO2–NH2 nanoprobes.
Fig. 1. TEM images of: (a) Fe3O4 nanoparticles and (b) Fe3O4(FITC)@SiO2–NH2 nanoprobes.
structure with an average size of 28 nm (Fig. 1b). The high uniformity of the as-synthesized nanoprobes could be attributed to the microemulsion method, which was effective for size and shape control of nanoparticles. The phase structures of the nanoparticles were investigated by X-ray diffraction (XRD). Fig. S3 illustrated XRD patterns of Fe3O4 nanoparticles and Fe3O4(FITC)@SiO2–NH2 nanaprobes. It was noted that the as-synthesized Fe3O4 nanoparticles (Fig. S3a) were well crystallized, and consistent with the cubic phase structure known from the bulk Fe3O4 crystal (JCPDS: 65-3107) with a space group of Fd-3m (2 2 7). In general, the nanocrystallite size can be estimated according to the Scherrer equation, D = 0.941k/b cos h, where D is the average grain size, k is the X-ray wavelength (0.15405 nm), and h and b are the diffraction angle and full-width at half-maximum (fwhm) of an observed peak, respectively [44]. The average crystallite sizes of Fe3O4 nanoparticles were determined to be 9.6 nm by using the strongest peak (3 1 1) at 2h = 35.6°, which was similar to the result of TEM (12 nm, Fig. 1a). In Fig. S3b, broad peak ranging from 20° to 30° was assigned to amorphous silica. Besides the broad peak of silica, the peaks of Fe3O4 nanoparticles were also observed in Fe3O4 (FITC)@SiO2–NH2 nanoprobes, revealing the introduction of the silica shell did not result in the phase change of Fe3O4 nanoparticles. The magnetic properties were investigated with a superconducting quantum interference device (SQUID) magnetometer under an applied magnetic field of 10,000 Oe. Fig. S4 showed the magnetization of the as-synthesized Fe3O4 nanoparticles and Fe3O4(FITC)@SiO2–NH2 nanoprobes measured at 300 K. Both of them exhibited negligible coercivity and remanence, typical of superparamagnetic materials, and their magnetic saturation values were determined
to be 30.6 and 2.4 emu (per gram sample), respectively. The obvious decrease in magnetization of Fe3O4(FITC)@SiO2–NH2 nanaprobes could be attributed to the presence of the thick shell of silica that is diamagnetic. The nanoprobes in water could be harvested by placing a NdFeB magnet close to the vial and redispersed into the solution after removing the magnet. The complete harvest of the nanoprobes to one inside wall of the vial was very quickly (15 min), which indicated the rapid response of the nanoprobes to external magnetic field. The fluorescence photograph of Fe3O4(FITC)@SiO2–NH2 nanoprobes was shown in Fig. 2a. The encapsulation of FITC dye in Fe3O4(FITC)@SiO2–NH2 nanoprobes endowed the nanoprobes with excellent fluorescent property. We observed that the nanoprobes emitted strong green fluorescence under a UV lamp (k = 365 nm). Furthermore, even after being harvested into one inside wall of the vial by a NdFeB magnet, the strong green fluorescence of the nanoprobes could be observed under UV excitation (Fig. 2b). The excitation and emission spectra of FITC dye in water were shown in Fig. S5a. The emission spectrum (Fig. S5a, solid line) of FITC dye includes a broad band ranging from 490 to 580 nm with a maximum at 517 nm. Monitored with the emission wavelength of 517 nm, the obtained excitation spectrum (Fig. S5a, dashed line) had a broad band ranging from 420 to 510 nm. The fluorescence photograph of FITC dye was shown in the inset of Fig. S5a. Its aqueous solution emitted green fluorescence under UV excitation in the dark. Fig. 3 illustrated the excitation and emission spectra of Fe3O4(FITC)@SiO2–NH2 nanoprobes. It was evident that Fe3O4 (FITC)@SiO2–NH2 nanoprobes and pure FITC dye had similar excitation and emission spectra, indicating the silica shell did not alter
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Fig. 2. Fluorescence photographs of Fe3O4(FITC)@SiO2–NH2 nanoprobes (a) in water and (b) in response to a NdFeB magnet under a UV lamp (k = 365 nm), respectively.
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nanoprobes were basically consistent with those of FITC dye-doped magnetic silica nanoparticles [28,29]. Furthermore, confocal laser scanning microscopy (CLSM) was used to investigate the fluorescence imaging of Fe3O4(FITC)@SiO2–NH2 nanoprobes (Fig. 4). The size of Fe3O4(FITC)@SiO2–NH2 nanoprobes in CLSM images (the droplet of the sample was placed between slide glass and coverglass) seems to be much larger than that in TEM images (the sample was dripped on copper grid and well dispersed), which is resulted from the two different method. Green fluorescence of the nanoprobes could be observed in fluorescence images (Fig. 4a), and the rough location and distribution of the nanoprobes can be observed in bright-field image (Fig. 4b). The results suggest the fluorescence originated from the nanoprobes themselves rather than the background. These attractive properties of Fe3O4(FITC)@SiO2–NH2 nanoprobes enabled the applications of MRI and cell fluorescence imaging. To verify this point, we firstly measured T2-relaxivity (R2) of the nanoprobes. The ability of proton relaxation enhancement of a paramagnetic compound as MRI contrast agent was commonly expressed by the term relaxivity. In our case, T2-relaxivity of the nanoprobes was determined to be 104.0 mM 1 s 1 at 25 °C (Fig. 5). Such a high relaxivity value may be due to the core–shell structure of Fe3O4(FITC)@SiO2–NH2 nanoprobes. Good solubility and excellent T2-relaxivity showed that the nanoprobes were very desirable for MRI application. We then proceeded to evaluate whether such nanoprobes could be applied to cell imaging (Fig. 6). In this case, HeLa cell was chosen as the model cell. These cells were cultured in DMEM medium, then incubated with Fe3O4 (FITC)@SiO2–NH2 nanoprobes. The green fluorescence of HeLa cells incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes was observed
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Wavelength (nm) Fig. 3. Excitation (dashed line, recorded at 522 nm emission) and emission (solid line, recorded at 470 nm excitation) spectra of Fe3O4(FITC)@SiO2–NH2 nanoprobes.
R2=104.0mM-1S-1 R2=0.997
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the spectra characteristics of FITC dye. The above spectral properties (excitation and emission spectra) of Fe3O4(FITC)@SiO2–NH2
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Fig. 4. CLSM images of Fe3O4(FITC)@SiO2–NH2 nanaprobes in: (a) fluorescence and (b) bright-field.
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in CLSM fluorescence images (Fig. 6a). Compared with the rough location and distribution of HeLa cell in bright-field images (Fig. 6b), we determine the fluorescence arises from the cell. To
verify the effect of the background to fluorescence, we use HeLa cells not incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes as control. There is no fluorescence signal in CLSM fluorescence image
Fig. 6. CLSM images of HeLa cells incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes (a and b) and not incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes (c and d) in fluorescence (a and c) and bright-field (b and d).
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Fig. 7. (a) TEM images, fluorescence photographs (b) in water and (c) in response to a NdFeB magnet under UV lamp (k = 365 nm) and (d) excitation (dashed line, recorded at 572 nm emission) and emission (solid line, recorded at 520 nm excitation) spectra of Fe3O4(RBITC)@SiO2–NH2 nanoprobes.
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(Fig. 6c), while the cell can be clearly observed in bright-field image (Fig. 6d). From the fluorescence images of HeLa cell incubated and not incubated with the nanoprobes, we can firmly conclude that the fluorescence should result from the nanoprobes rather than from background or HeLa cells. Endocytosis is the main approach of cellular internalization for nanoparticles. Fluorescence intensity can be utilized to determine semiquantitatively the cellular uptake. Namely, the stronger of fluorescence of the cells was, the more of the nanoprobes were endocytosed. The results of CLSM images showed the successful application of cell fluorescence imaging of Fe3O4(FITC)@SiO2–NH2 nanoprobes. It is fully believed that fluorescent magnetic Fe3O4(FITC)@SiO2–NH2 nanoprobes allow the potential applications of dual imaging in biomedical field. To verify that this strategy can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group, we prepared Fe3O4(RBITC)@SiO2–NH2 nanoprobes under identical conditions. Rhodamine B isothiocyanate–3-aminopropyltriethoxysilane (RBITC–APTES) conjugates were prepared in advance via an addition reaction between isothiocyanate group of RBITC dye and primary amines of APTES. TEM images show that Fe3O4(RBITC)@SiO2–NH2 nanoprobes are uniformly spherical core– shell structure with size of 28 nm (Fig. 7a). The fluorescence photograph of Fe3O4(RBITC)@SiO2–NH2 nanoprobes was shown in Fig. 7b. The aqueous solution emitted strong red fluorescence under UV excitation (k = 365 nm). The nanoprobes could be harvested by placing a NdFeB magnet close to the vial (Fig. 7c) and the complete harvest of the nanoprobes only took 15 min, which indicated the rapid response of the nanoprobes to external magnetic field. The excitation and emission spectra of Fe3O4(RBITC)@SiO2–NH2 nanoprobes in water were demonstrated in Fig. 7d, which were similar to those of pure RBITC in water (Fig. S5b). Fe3O4(RBITC)@SiO2–NH2 nanoprobes might be suitable for T2-relaxivity and cell fluorescence imaging. 4. Conclusions We report the synthesis of fluorescent magnetic Fe3O4(FITC)@SiO2–NH2 and Fe3O4(RBITC)@SiO2–NH2 nanoprobes, and demonstrate the T2-relaxtivity and the application for cell fluorescence imaging of Fe3O4(FITC)@SiO2–NH2 nanoprobes. The particularly attractive features of our as-synthesized nanoprobes are as follows: (1) The dyes in the nanoprobes can keep their photochemical stability because they are isolated from external environment via silica shell. (2) The primary amines of silica surface provide a platform to graft other functional and targeted moieties depending on the requirements. (3) The nanoprobes can act as promising labeling materials due to their excellent magnetic and fluorescent properties. (4) The nanoprobes are highly water-soluble and biocompatible which make them bear potential application in biomedical field. (5) The synthetic method can be extended to synthesize other bifunctional nanoprobes by using the dye with isothiocyanate group. Acknowledgments Financial support by the Program for Excellent Doctoral Thesis of Chinese Academy of Sciences, ‘‘Hundred Talents Project” of Chinese Academy of Sciences, and the National Basic Research Program of China (973 Program; No. 2010CB933600), NSFC (No. 20873138) is gratefully acknowledged. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2010.07.056.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Fluorescent magnetic nanoprobes: Design and application for cell imaging Guo Zhang a,c, Jianghua Feng b,**, Lehui Lu a,*, Baohua Zhang a,c, Linyuan Cao a,c a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China State Key Laboratory of Magnetic Resonance and Molecular and Atomic Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, Hubei 430071, PR China c Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China b
a r t i c l e
i n f o
Article history: Received 31 May 2010 Accepted 21 July 2010 Available online 27 July 2010 Keywords: Nanoprobes Magnetic Fluorescent T2-relaxivity Cell imaging
a b s t r a c t Multifunctional nanoprobes combining magnetic nanoparticles with organic dyes have attracted tremendous interest due to their promising applications in biomedical field. Here we demonstrate a facile and general strategy for the fabrication of robust fluorescent magnetic nanoprobes with high payloads of dye molecules and their use as multimodal nanoprobes for cell imaging. These nanoprobes not only effectively keep photochemical stability of dyes, but also provide a platform for grafting other functional or targeted moieties into silica surface via primary amines. Moreover, the nanoprobes are uniformly spherical morphology and can be dispersed well in aqueous solution, which are very desirable for biomedical applications. Importantly, this method can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Magnetic resonance imaging (MRI) is one of the most powerful and non-invasive imaging techniques by measuring proton relaxation processes of water in biological systems. In order to meet the different demands of biomedical research, Fe3O4 nanoparticles [1– 6], as MRI contrast enhancement agents [7–10], have been employed as a core platform to construct multimodal probes by the conjugation with other functional moieties including optical tags, radionuclides, targeting moiety, and drugs or gene carriers [11– 15]. Among these nanoprobes, Fe3O4 nanoparticles-based MRIfluorescent nanoprobes are of growing interest [16–19]. These MRI-fluorescent nanoprobes not only bridge gaps in sensitivity and depth of imaging between the two modalities, but also simultaneously obtain multiple imaging information data. Fluorescent molecules such as organic dyes could be directly conjugated to magnetic nanoparticles to produce MRI-fluorescent nanoprobes. These nanoprobes work nicely, but have the potential adversities including photobleaching and rapid fluorescence quenching effects [20]. To improve the stability and versatility of the fluorescent magnetic nanoprobes, desirable surface coating materials (e.g. polymer, dextran, etc.) or functional molecules with specific targeting groups (e.g. antibodies, peptides, etc.) are required. Also, silica has already served as candidate to encapsulate nanoprobes
* Corresponding author. Fax: +86 431 85262406. ** Corresponding author. E-mail addresses: [email protected] (J. Feng), [email protected] (L. Lu). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.07.056
because of its stability, biocompatibility, easy functionalization, and low cytotoxicity [21–27]. Silica-coated fluorescent magnetic nanoprobes have been recently reported by many research groups [28–35]. These diverse nanoprobes allow the development of biomedical platforms for simultaneous imaging, diagnosis and therapy. However, it is challenging to fabricate fluorescent magnetic nanoprobes with high photostability, high payloads of dyes and desirable outer surface for further modifications with functional or target moieties. To address the above issues, we put forward a design strategy of fluorescent magnetic nanoprobes. This strategy is facile and general for the synthesis of multifunctional nanoprobes combining magnetic Fe3O4 nanoparticles and dyes with isothiocyanate group. The as-synthesized nanoprobes including Fe3O4(FITC)@SiO2–NH2 and Fe3O4(RBITC)@SiO2–NH2 possess excellent fluorescent and magnetic properties, and good water solubility. Investigation on T2-relaxivity and cell fluorescence imaging of Fe3O4(RBITC)@SiO2–NH2 nanoprobes indicates that these nanoprobes are desirable labeling materials and may find potential applications in biomedical field. 2. Material and methods 2.1. Materials All chemicals were of analytic grade and used without further purification unless indicated. 1-Octadecene (ODE, 90%), oleic acid (90%), APTES, RBITC, polyoxyethylene (5) nonylphenyl ether (Igepal CO-520), ammonia solution (28%) and TEOS were
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purchased from Aldrich. FITC was purchased from Fluka. The water used was purified through a Millipore system. 2.2. Synthesis of Fe3O4 nanoparticles Monodisperse 12-nm Fe3O4 nanoparticles were synthesized by high-temperature thermal decomposition iron–oleate complex [36]. Firstly, iron–oleate complex was prepared by a reaction of ferric chloride (FeCl36H2O, 1.08 g, 4 mmol) and sodium oleate (3.65 g, 12 mmol) in a mixture solvent composed ethanol (8 mL), water (6 mL) and hexane (14 mL) at 70 °C for 4 h. Next, the upper organic layer containing iron–oleate complex was washed three times with water (10 mL) in a separatory funnel after completing the reaction, resulting in iron–oleate complex in a waxy solid form. The as-synthesized iron–oleate complex (3.6 g, 4 mmol) and oleic acid (0.57 g, 2 mmol) were dissolved in ODE (20 g) at room temperature and were heated to 320 °C with a heating rate of 3.3 °C min 1, and then kept for 30 min. After cooling to room temperature, the resulting Fe3O4 nanoparticles were precipitated by adding ethanol (50 mL), separated by centrifugation, and finally dried by vacuum at 80 °C. 2.3. Synthesis of FITC–APTES (or RBITC–APTES) conjugates FITC (5 mg) was conjugated with APTES (10 lL) in anhydrous ethanol (3 mL) by an addition reaction of isothiocyanate group with amine group. The reaction was carried out in dark for 12 h by slowly stirring under argon atmosphere. The reaction solution of FITC–APTES conjugates was then stored at 4 °C. Under identical conditions, RBITC–APTES conjugates were synthesized by an addition reaction of RBITC (13.8 mg) with APTES (10 lL) in anhydrous ethanol (3 mL) and stored at 4 °C. 2.4. Synthesis of Fe3O4(FITC)@SiO2–NH2 nanoprobes Fe3O4(FITC)@SiO2–NH2 nanoprobes were prepared by an improved microemulsion method [37]. Briefly, Igepal CO-520 (0.46 mL, 1 mmol) was dispersed into cyclohexane (9 mL) in a 50-mL three-necked flask by ultrasonication for 20 min. Under mechanical stirring, Fe3O4 cyclohexane solution (1 mg mL 1, 1 mL) and FITC–APTES conjugates (20 lL) were added into the microemulsion. Then, ammonia solution (80 lL) and TEOS (60 lL) was added, the mixture was allowed to age for 24 h to hydrolysis and condensation of the silica precursor. The final surface layer incorporating primary amines was formed by adding APTES (0.15 lL) and stirring for 24 h. The resulting Fe3O4(FITC)@SiO2–NH2 nanoprobes were collected by centrifugation and finally washed with ethanol three times and water three times, respectively. Similarly, using RBITC–APTES conjugates (20 lL) instead of FITC–APTES conjugates, Fe3O4(RBITC)@SiO2–NH2 can be made. 2.5. The measurement of T2-relaxivity (R2) Fe3O4(FITC)@SiO2–NH2 nanoparticles were dispersed in deuterated water (D2O). Five samples were prepared separately with the iron concentration of 0, 0.05, 0.1, 0.25, and 0.5 mM. Their transverse relaxation time (T2) was measured by using Carr–Purcell– Meiboom–Gill (CPMG) pulse sequence at 11.7 T and 25 °C using an NMR spectrometer. The relaxivities (R2) were obtained from the linear regressions (the relaxation rates (1/T2) vs. iron concentration). 2.6. Cell-uptake of Fe3O4(FITC)@SiO2–NH2 nanoprobes HeLa cells were seeded at a density of 6.0 104 cells/3 mL in a 6-well plate containing coverslips. Cell culture was maintained at
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37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) medium for 24 h. To determine cell-uptake of Fe3O4(FITC)@SiO2– NH2 nanoprobes, the cells were incubated with 10 lg mL 1 (the concentration of Fe) Fe3O4(FITC)@SiO2–NH2 nanoprobes in phosphate buffered saline (PBS) dispersion solution at 37 °C for 3 h. After washing with warm of PBS solution (2 mL), the cell sample was soaked into 4% PFA (para-formaldehyde) for 15 min. Following this, sample slips were washed with PBS and H2O several times, then dried. Finally, coverslips were mounted on drop of neutral balsam, leaving to dry at room temperature in the dark for confocal microscopy. 2.7. Characterization TEM images were obtained on an H-8100 TEM operating at 200 kV. XRD of the powder samples were examined on a D8 Focus diffractometer (Bruker) using Cu Ka radiation (k = 0.15405 nm). Magnetic studies were carried out on a Quantum Design SQUID magnetometer. Fluorescence photographs were taken on a Canon digital camera. The excitation and emission spectra were collected on an F-4500 spectrophotometer equipped with a 150 W xenon lamp as the excitation source. CLSM images were obtained on a CLSM (Leica TCS SP2). T2-relaxivity was measured at 11.7 T using an NMR spectrometer (Bruker AVII-500). 3. Results and discussion The synthetic process of the fluorescent magnetic Fe3O4(FITC)@SiO2–NH2 nanoprobes was shown in Scheme 1. Typically, the as-synthesized Fe3O4 dispersion and fluorescein isothiocyanate–3-aminopropyltriethoxysilane (FITC–APTES) conjugates were simultaneously added into a microemulsion, and APTES was added into the above microemulsion after 24 h. FITC–APTES conjugates were prepared in advance via an addition reaction between the isothiocyanate group of FITC dye and the primary amines group of APTES. This synthetic process enables the co-encapsulation of Fe3O4 nanoparticles and large amount of FITC dye molecules together via silica shell (Fig. S1a), and the grafting of primary amines into the silica surface. Such a core–shell structure has some advantages. First, it can prevent fluorescence quenching of the nanoprobes because FITC dye is not directly conjugated to Fe3O4 nanoparticles via chemical reaction (Fig. S1b) [38]; second, it can keep photochemical stability and increase fluorescence intensity of the dyes due to ‘‘caging effects” of silica shell [39–43]; third, it bears the potential to facilitate surface modification via primary amines that are more active than silanol groups. The amine site is an important surface-bound moiety to graft specific functional groups and biomolecules for biomedicine applications. The as-synthesized nanoprobes can be well-dispersed in aqueous solution, thus make them feasible to measure relaxivity and run the cell experiments. Transmission electron microscopy (TEM) images of Fe3O4 nanoparticles and Fe3O4(FITC)@SiO2–NH2 nanoprobes were shown in Fig. 1. Fe3O4 nanoparticles prepared via high-temperature thermal decomposition method were monodisperse with an average diameter of 12 nm (Fig. 1a). A thick layer of silica shell was then formed in situ through hydrolysis of tetraethyl orthosilicate (TEOS) and subsequent condensation of silica onto the surface of Fe3O4 cores, which was determined to be 8 nm. The thickness of the silica shell can be tuned by varying the volume of TEOS. A thick silica shell was preferred in this case since a thicker silica shell allowed high payloads of dye molecules to be achieved (Fig. S2). The final surface layer incorporating primary amines was formed in situ through hydrolysis of APTES. TEM image of as-synthesized Fe3O4(FITC)@SiO2–NH2 nanoprobes showed well-defined spherical core–shell
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Scheme 1. Schematic representation of the synthetic process, structure and property of Fe3O4(FITC)@SiO2–NH2 nanoprobes.
Fig. 1. TEM images of: (a) Fe3O4 nanoparticles and (b) Fe3O4(FITC)@SiO2–NH2 nanoprobes.
structure with an average size of 28 nm (Fig. 1b). The high uniformity of the as-synthesized nanoprobes could be attributed to the microemulsion method, which was effective for size and shape control of nanoparticles. The phase structures of the nanoparticles were investigated by X-ray diffraction (XRD). Fig. S3 illustrated XRD patterns of Fe3O4 nanoparticles and Fe3O4(FITC)@SiO2–NH2 nanaprobes. It was noted that the as-synthesized Fe3O4 nanoparticles (Fig. S3a) were well crystallized, and consistent with the cubic phase structure known from the bulk Fe3O4 crystal (JCPDS: 65-3107) with a space group of Fd-3m (2 2 7). In general, the nanocrystallite size can be estimated according to the Scherrer equation, D = 0.941k/b cos h, where D is the average grain size, k is the X-ray wavelength (0.15405 nm), and h and b are the diffraction angle and full-width at half-maximum (fwhm) of an observed peak, respectively [44]. The average crystallite sizes of Fe3O4 nanoparticles were determined to be 9.6 nm by using the strongest peak (3 1 1) at 2h = 35.6°, which was similar to the result of TEM (12 nm, Fig. 1a). In Fig. S3b, broad peak ranging from 20° to 30° was assigned to amorphous silica. Besides the broad peak of silica, the peaks of Fe3O4 nanoparticles were also observed in Fe3O4 (FITC)@SiO2–NH2 nanoprobes, revealing the introduction of the silica shell did not result in the phase change of Fe3O4 nanoparticles. The magnetic properties were investigated with a superconducting quantum interference device (SQUID) magnetometer under an applied magnetic field of 10,000 Oe. Fig. S4 showed the magnetization of the as-synthesized Fe3O4 nanoparticles and Fe3O4(FITC)@SiO2–NH2 nanoprobes measured at 300 K. Both of them exhibited negligible coercivity and remanence, typical of superparamagnetic materials, and their magnetic saturation values were determined
to be 30.6 and 2.4 emu (per gram sample), respectively. The obvious decrease in magnetization of Fe3O4(FITC)@SiO2–NH2 nanaprobes could be attributed to the presence of the thick shell of silica that is diamagnetic. The nanoprobes in water could be harvested by placing a NdFeB magnet close to the vial and redispersed into the solution after removing the magnet. The complete harvest of the nanoprobes to one inside wall of the vial was very quickly (15 min), which indicated the rapid response of the nanoprobes to external magnetic field. The fluorescence photograph of Fe3O4(FITC)@SiO2–NH2 nanoprobes was shown in Fig. 2a. The encapsulation of FITC dye in Fe3O4(FITC)@SiO2–NH2 nanoprobes endowed the nanoprobes with excellent fluorescent property. We observed that the nanoprobes emitted strong green fluorescence under a UV lamp (k = 365 nm). Furthermore, even after being harvested into one inside wall of the vial by a NdFeB magnet, the strong green fluorescence of the nanoprobes could be observed under UV excitation (Fig. 2b). The excitation and emission spectra of FITC dye in water were shown in Fig. S5a. The emission spectrum (Fig. S5a, solid line) of FITC dye includes a broad band ranging from 490 to 580 nm with a maximum at 517 nm. Monitored with the emission wavelength of 517 nm, the obtained excitation spectrum (Fig. S5a, dashed line) had a broad band ranging from 420 to 510 nm. The fluorescence photograph of FITC dye was shown in the inset of Fig. S5a. Its aqueous solution emitted green fluorescence under UV excitation in the dark. Fig. 3 illustrated the excitation and emission spectra of Fe3O4(FITC)@SiO2–NH2 nanoprobes. It was evident that Fe3O4 (FITC)@SiO2–NH2 nanoprobes and pure FITC dye had similar excitation and emission spectra, indicating the silica shell did not alter
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Fig. 2. Fluorescence photographs of Fe3O4(FITC)@SiO2–NH2 nanoprobes (a) in water and (b) in response to a NdFeB magnet under a UV lamp (k = 365 nm), respectively.
0.8 0.6
60
0.4
50
0.2
40
1/T2(s-1)
Intensity (normalized)
1.0
nanoprobes were basically consistent with those of FITC dye-doped magnetic silica nanoparticles [28,29]. Furthermore, confocal laser scanning microscopy (CLSM) was used to investigate the fluorescence imaging of Fe3O4(FITC)@SiO2–NH2 nanoprobes (Fig. 4). The size of Fe3O4(FITC)@SiO2–NH2 nanoprobes in CLSM images (the droplet of the sample was placed between slide glass and coverglass) seems to be much larger than that in TEM images (the sample was dripped on copper grid and well dispersed), which is resulted from the two different method. Green fluorescence of the nanoprobes could be observed in fluorescence images (Fig. 4a), and the rough location and distribution of the nanoprobes can be observed in bright-field image (Fig. 4b). The results suggest the fluorescence originated from the nanoprobes themselves rather than the background. These attractive properties of Fe3O4(FITC)@SiO2–NH2 nanoprobes enabled the applications of MRI and cell fluorescence imaging. To verify this point, we firstly measured T2-relaxivity (R2) of the nanoprobes. The ability of proton relaxation enhancement of a paramagnetic compound as MRI contrast agent was commonly expressed by the term relaxivity. In our case, T2-relaxivity of the nanoprobes was determined to be 104.0 mM 1 s 1 at 25 °C (Fig. 5). Such a high relaxivity value may be due to the core–shell structure of Fe3O4(FITC)@SiO2–NH2 nanoprobes. Good solubility and excellent T2-relaxivity showed that the nanoprobes were very desirable for MRI application. We then proceeded to evaluate whether such nanoprobes could be applied to cell imaging (Fig. 6). In this case, HeLa cell was chosen as the model cell. These cells were cultured in DMEM medium, then incubated with Fe3O4 (FITC)@SiO2–NH2 nanoprobes. The green fluorescence of HeLa cells incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes was observed
0.0 420 440 460 480 500 520 540 560 580 600
Wavelength (nm) Fig. 3. Excitation (dashed line, recorded at 522 nm emission) and emission (solid line, recorded at 470 nm excitation) spectra of Fe3O4(FITC)@SiO2–NH2 nanoprobes.
R2=104.0mM-1S-1 R2=0.997
30 20 10 0 0.0
the spectra characteristics of FITC dye. The above spectral properties (excitation and emission spectra) of Fe3O4(FITC)@SiO2–NH2
0.1
0.2
0.3
0.4
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Fe concentration (mM) Fig. 5. T2-relaxivity plot of Fe3O4(FITC)@SiO2–NH2 nanoprobes.
Fig. 4. CLSM images of Fe3O4(FITC)@SiO2–NH2 nanaprobes in: (a) fluorescence and (b) bright-field.
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in CLSM fluorescence images (Fig. 6a). Compared with the rough location and distribution of HeLa cell in bright-field images (Fig. 6b), we determine the fluorescence arises from the cell. To
verify the effect of the background to fluorescence, we use HeLa cells not incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes as control. There is no fluorescence signal in CLSM fluorescence image
Fig. 6. CLSM images of HeLa cells incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes (a and b) and not incubated with Fe3O4(FITC)@SiO2–NH2 nanoprobes (c and d) in fluorescence (a and c) and bright-field (b and d).
Intensity (normalized)
1.0
d
0.8 0.6 0.4 0.2 0.0 420 440 460 480 500 520 540 560 580 600 620 640
Wavelength (nm)
Fig. 7. (a) TEM images, fluorescence photographs (b) in water and (c) in response to a NdFeB magnet under UV lamp (k = 365 nm) and (d) excitation (dashed line, recorded at 572 nm emission) and emission (solid line, recorded at 520 nm excitation) spectra of Fe3O4(RBITC)@SiO2–NH2 nanoprobes.
G. Zhang et al. / Journal of Colloid and Interface Science 351 (2010) 128–133
(Fig. 6c), while the cell can be clearly observed in bright-field image (Fig. 6d). From the fluorescence images of HeLa cell incubated and not incubated with the nanoprobes, we can firmly conclude that the fluorescence should result from the nanoprobes rather than from background or HeLa cells. Endocytosis is the main approach of cellular internalization for nanoparticles. Fluorescence intensity can be utilized to determine semiquantitatively the cellular uptake. Namely, the stronger of fluorescence of the cells was, the more of the nanoprobes were endocytosed. The results of CLSM images showed the successful application of cell fluorescence imaging of Fe3O4(FITC)@SiO2–NH2 nanoprobes. It is fully believed that fluorescent magnetic Fe3O4(FITC)@SiO2–NH2 nanoprobes allow the potential applications of dual imaging in biomedical field. To verify that this strategy can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group, we prepared Fe3O4(RBITC)@SiO2–NH2 nanoprobes under identical conditions. Rhodamine B isothiocyanate–3-aminopropyltriethoxysilane (RBITC–APTES) conjugates were prepared in advance via an addition reaction between isothiocyanate group of RBITC dye and primary amines of APTES. TEM images show that Fe3O4(RBITC)@SiO2–NH2 nanoprobes are uniformly spherical core– shell structure with size of 28 nm (Fig. 7a). The fluorescence photograph of Fe3O4(RBITC)@SiO2–NH2 nanoprobes was shown in Fig. 7b. The aqueous solution emitted strong red fluorescence under UV excitation (k = 365 nm). The nanoprobes could be harvested by placing a NdFeB magnet close to the vial (Fig. 7c) and the complete harvest of the nanoprobes only took 15 min, which indicated the rapid response of the nanoprobes to external magnetic field. The excitation and emission spectra of Fe3O4(RBITC)@SiO2–NH2 nanoprobes in water were demonstrated in Fig. 7d, which were similar to those of pure RBITC in water (Fig. S5b). Fe3O4(RBITC)@SiO2–NH2 nanoprobes might be suitable for T2-relaxivity and cell fluorescence imaging. 4. Conclusions We report the synthesis of fluorescent magnetic Fe3O4(FITC)@SiO2–NH2 and Fe3O4(RBITC)@SiO2–NH2 nanoprobes, and demonstrate the T2-relaxtivity and the application for cell fluorescence imaging of Fe3O4(FITC)@SiO2–NH2 nanoprobes. The particularly attractive features of our as-synthesized nanoprobes are as follows: (1) The dyes in the nanoprobes can keep their photochemical stability because they are isolated from external environment via silica shell. (2) The primary amines of silica surface provide a platform to graft other functional and targeted moieties depending on the requirements. (3) The nanoprobes can act as promising labeling materials due to their excellent magnetic and fluorescent properties. (4) The nanoprobes are highly water-soluble and biocompatible which make them bear potential application in biomedical field. (5) The synthetic method can be extended to synthesize other bifunctional nanoprobes by using the dye with isothiocyanate group. Acknowledgments Financial support by the Program for Excellent Doctoral Thesis of Chinese Academy of Sciences, ‘‘Hundred Talents Project” of Chinese Academy of Sciences, and the National Basic Research Program of China (973 Program; No. 2010CB933600), NSFC (No. 20873138) is gratefully acknowledged. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2010.07.056.
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