Synthesis of Water-Dispersible Mn2+ Functionalized Silicon

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Synthesis of Water-Dispersible Mn2+ Functionalized Silicon Nanoparticles under Room Temperature and Atmospheric Pressure for Fluorescence and Magnetic Resonance Dual-Modality Imaging Ya-Kun Dou, Yang Chen, Xi-Wen He, Wen-You Li, Yu-Hao Li, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01644 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Analytical Chemistry

Synthesis of Water-Dispersible Mn2+ Functionalized Silicon Nanoparticles under Room Temperature and Atmospheric Pressure for Fluorescence and Magnetic Resonance Dual-Modality Imaging Ya-Kun Dou,† Yang Chen,‡ Xi-Wen He,† Wen-You Li,*,†,§Yu-Hao Li,*,‡ and Yu-Kui Zhang†,

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†College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China, [email protected]., Fax: +86-22-23502458 ‡Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Nankai University School of Medicine, Tianjin 300071, China, [email protected]., Fax: +86-22-23502554 §Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China ‖National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China ABSTRACT: Silicon nanoparticles (Si NPs) have been widely used in fluorescence imaging. However, rigorous synthesis conditions and the single modality imaging limit the further development of Si NPs in the field of biomedical imaging. Here, we reported a method for synthesizing water-dispersible Mn2+ functionalized Si NPs (Mn-Si NPs) under mild experimental conditions for fluorescence and magnetic resonance dual-modality imaging. The whole synthesis process was completed under room temperature and atmospheric pressure, and no special and expensive equipment was required. The synthetic nanoparticles with favorable pH stability, NaCl stability, photostability and low toxicity, emitted green fluorescence (512 nm). At the same time, the nanoparticles also demonstrated excellent magnetic resonance imaging ability. In vitro their T1-weighted magnetic resonance imaging effect was obvious and the value of longitudinal relaxation degree r1 reached 4.25 mM-1 s-1. Based on their good biocompatibility, Mn-Si NPs were successfully used for the fluorescence imaging as well as magnetic resonance imaging in vivo.

In recent years, semiconductor nanoparticles have been attracted more and more attention in the field of biomedical imaging.1-4 Nevertheless, the toxicity hinders the further development of the nanoparticles in biological field.57 Hence, the silicon nanoparticles (Si NPs) with lower toxicity have emerged as the times require. Silicon nanoparticles have many excellent properties, such as abundant reserves of silicon, good water solubility, strong photostability, low toxicity and favorable biocompatibility,8-10 which make them have good application prospects in biological fluorescence imaging. So far, many methods to synthesize Si NPs were reported, such as electrochemical etching silicon wafers, 11-15 laser-driven pyrolysis of silanes,16-21 microwave-assisted synthesis,22-25 and so on. Nevertheless, these synthetic methods often need harsh synthesis conditions (e.g., high temperature: generally higher than 150 °C, hydrofluoric acid and nitric acid etching) or special instruments and equipment (e.g., microwave reactor, plasma reactor, etc.). Therefore, it is a challenge to synthesize silicon nanoparticles under mild conditions. To overcome these difficulties, many efforts have been made. Recently, Wang et al.26 developed a method for

synthesizing silicon nanoparticles under room temperature and atmospheric pressure and applied them to cell fluorescence imaging. In our group,27 silicon nanoparticles were synthesized via the method of microwave assisted synthesis at atmospheric pressure and applied to fluorescence imaging of cells and zebrafish. Howbeit, the synthetic nanoparticles were only applied to the single modality fluorescence imaging. As we all know, the single modality imaging does not provide comprehensive information for diagnosis and treatment.28 Hence, multimodal imaging technologies which can provide more comprehensive and accurate information on diagnosis have received much attention in the field of biomedical applications.29,30 Among them, the magnetic resonance imaging (MRI) technology has been widely applied in the field of biological imaging because of its high spatial resolution.3133 Mn2+ has strong paramagnetism because of its third orbit having 5 unpaired electrons. Furthermore, manganese is one of the essential trace elements for normal body and it plays an important role in maintaining the normal physiological function of human body. These advantages make Mn2+ a good magnetic resonance imaging agent in vivo. Mn2+ doped nanoparticles (ZnS:Mn2+, ZnSe:Mn2+,

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CdSe:Mn2+ QDs) have been used in magnetic resonance imaging.34 Recently, McVey et al.35 used LiAlH4 as reductant to synthesize Mn2+ doped silicon nanoparticles in anhydrous and anaerobic environment. Kauzlarich’s group 36,37 used NaH as reductant to prepare Mn2+ doped silicon nanoparticles under the condition of 500 °C high temperature and in anhydrous and anaerobic environment. However, these methods all required the presence of dangerous reductant or high temperature, which increased the requirements for the experimental equipment and raised the experimental risk. Consequently, it is necessary to synthesize a novel Mn2+ functionalized silicon nanoparticles with both fluorescence and magnetic resonance imaging capability under mild experimental conditions. Here, we reported a method for synthesizing waterdispersible Mn2+ functionalized Si NPs (Mn-Si NPs) as fluorescence and MR dual-modality imaging probe under mild experimental conditions. The whole synthesis process was carried out under room temperature and atmospheric pressure, and did not require any expensive and special equipment. The synthetic Mn-Si NPs not only retained excellent fluorescence properties, but also had fine magnetic resonance response. Based on the low toxicity of the as-prepared Mn-Si NPs, we successfully applied the nanoparticles as a dual-modality probe to image in vitro and in vivo. EXPERIMENTAL SECTION Synthesis of the Silicon Nanoparticles (Si NPs). Add 5mL water to 25 mL round bottom flask, 1 mL N-(2aminoethyl)-3-aminopropyltriethoxysilane (N-APTES, 98%+) was added and stirred 5 min, and then add 3 mL 0.1 M (+)-sodium L-ascorbate (AS, freshly prepared) into the flask and stirred 25 min, getting the silicon nanoparticles (Si NPs). Activation of EDTA-MnNa2. 0.6610 g EDTA-MnNa2 was dissolved into 7 mL water, and adjusted pH 5.0 with HCl, then 0.3350 g EDC and 0.5035 g NHS was added to activate EDTA-MnNa2 with stirring for 30min. Preparation of Mn-Si NPs. The silicon nanoparticles were added to the activated EDTA-MnNa2, reacting 12 h under the room temperature (25 °C) and atmospheric pressure. Excess reagent could be removed from Mn-Si NPs products by dialyzing through a dialysis membrane (1000 MW). The as-prepared Mn-Si NPs were stored in the dark and the solid product was obtained after freezedrying for further applications. ICP-AES results showed that the Si content in Mn-Si NPs was 1.83 mg/mL and the content of Mn2+ in Mn-Si NPs was 0.50 mg/mL. It was worth noting that the concentration of Mn-Si NPs involved in fluorescence imaging is the content of silicon. Other experimental sections, including chemicals and reagents, instrumentations, cytotoxicity assay, imaging experiment, etc., were described in detail in Supporting Information. RESULTS AND DISCUSSION

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The Optimization of Reaction Conditions. Scheme 1 shows the schematic illustration for the preparation of water-dispersible Mn2+ functionalized Si NPs under room temperature and atmospheric pressure. First, we synthesized Si NPs according to Chen’s method. 26 At the same time, EDTA-MnNa2 was activated in the presence of EDC and NHS. Then the activated EDTA-MnNa2 was coupled onto the Si NPs via EDC and NHS coupling reaction to get Mn-Si NPs. We optimized the synthetic reaction conditions in order to obtain the best fluorescence properties of Mn-Si NPs. The conditions included three aspects: the molar ratios of N-APTES to AS, N-APTES to EDTA-MnNa2, and EDC to NHS. Figure S1 showed the optimization results. As shown in Figure S1a and S1b, the PL intensity changed with the various molar ratios of N-APTES to AS. The PL intensity obviously increased along with the increase of the ratio until 1:0.084 and slightly decreased as the ratio further increased. The emission wavelength did not change with the various molar ratios. Figure S1c and S1d described the influences of the molar ratios of NAPTES to EDTA-MnNa2 on the PL intensity and emission wavelength. The maximum PL intensity was obtained when the ratio was 1:0.5. The emission wavelength was basically consistent at the ratio of 1:0.125 to 1:0.625 and decreased remarkably as the ratio further increased. As presented in Figure S1e and S1f, the PL intensity and emission wavelength varied with the molar ratios of EDC to NHS. As the ratio changed from 1:0 to 1:6, the PL intensity and emission wavelength first increased and reached a maximum when the molar ratios of EDC to NHS was 1:2.5 and then decreased. In summary, Mn-Si NPs were synthesized by stirring various substances with the molar ratios of 1:0.084 (N-APTES to AS), 1:0.5 (N-APTES to EDTAMnNa2) and 1:2.5 (EDC to NHS) under room temperature and atmospheric pressure for 12 h. The Structure Characterizations of Mn-Si NPs. Figure 1a showed the transmission electron microscope (TEM) and the high resolution transmission electron microscopy (HRTEM) images of Mn-Si NPs. The as-prepared Mn-Si NPs showed good monodispersity with spherical shape and good size uniformity. As shown in the inset in Figure 1a, the lattice spacing of the as-prepared Mn-Si NPs was 0.32 nm, agreeing with the lattice of Si (111), which was consistent with the reported literature.26 The average hydrodynamic diameter of the as-prepared Mn-Si NPs measured by dynamic light scattering (DLS) was 5.4 nm as shown in Figure 1b. In order to certify the successful coupling between Si NPs and EDTA-MnNa2, we measured the FTIR of Si NPs and Mn-Si NPs as shown in Figure 1c. The broad absorbance peaks at 3360 cm−1 and the absorbance peaks at 1598 cm−1 were respectively attributed to the stretching vibration and bending vibration of the N−H bond. Typically, the sharp absorbance peaks at ∼1120-1030cm−1 were ascribed to the vibrational stretch of Si−O−Si bonding which was consistent with the previously reported results.38 And the absorbance peaks at 2932, 2878 and 2880 cm−1 were corresponded to the vibrational stretch of –CH2 bond. These absorbance peaks existed both in Si NPs and


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Scheme 1. The schematic illustration of Mn-Si NPs synthesis.

Figure 1. The structure characterizations of Mn-Si NPs. (a) TEM. Inset: photographs of HRTEM. (b) DLS. (c) FTIR spectra. (d) The zeta potential of Si NPs and Mn-Si NPs at neutral pH. Mn-Si NPs. However, there are some absorption peaks that only exist in Mn-Si NPs. Such as the bending vibration of N-H was focused on 713 cm-1, the absorbance peak at 1655 cm-1 was attributed to the stretching vibration of C=O and 1260 cm-1 and 645 cm-1 were the stretching vibration of C-N and the bending vibration of O=C-N, respectively. These absorbance peaks proved the generation of the O=C-N bond, as well as the success of the coupling. The zeta potential of Si NPs and Mn-Si NPs at neutral pH

were 11.67±1.58 and 3.68±0.33 mV, respectively as shown in Figure 1d. The changes of zeta potential before and after coupling of silicon nanoparticles proved the success of the coupling. 26,30,39 And the surface components of Mn-Si NPs determined by the XPS (Figure S2) were in good agreement with FTIR results. The Optical Properties of Mn-Si NPs. The optical absorption and fluorescence spectra of Mn-Si NPs are shown in Figure S3 and Figure 2. As shown in Figure S3, there



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were obvious differences between the nanoparticles before and after coupling with EDTA-MnNa2. Silicon nanoparticles before coupling emitted blue fluorescence (462nm) (black line in Figure S3). However, the emission wavelength of silicon nanoparticles after coupling was red-shifted to 512nm, and the luminescent intensity was higher (red line in Figure S3). The results proved the success of the coupling. In our opinion, the different sizes between Si NPs and Mn-Si NPs are the reason for the redshift.7,40-41 As shown in Figure 1 and Figure S4 , from the TEM, we found that the average size of Si NPs was 3.2 nm (Figure S4a), while Mn-Si NPs’ average size was 4.3 nm (Figure 1a). We also found that the average hydrodynamic diameter of Si NPs and as-prepared Mn-Si NPs measured by dynamic light scattering (DLS) was 4.1 nm (Figure S4b)

Figure 2. UV−vis absorption spectrum (black line), fluorescence excitation spectrum (blue line) and PL spectrum (red line) of Mn-Si NPs. Inset: photographs under natural light (left) and 365 nm UV lamp irradiation (right) of Mn-Si NPs.

Figure 3. Viability of A549 cells after incubation with different concentrations (0, 200, 300 and 500 μg/mL) of MnSi NPs for 3 h, 12 h and 24 h. and 5.4 nm (Figure 1b), respectively. From these results we can see that the size of as-prepared Mn-Si NPs was bigger than that of Si NPs, which resulted in the red-shift of MnSi NPs. 7,40-41 Broad absorption was observed for the wavelength region < 500 nm for Mn-Si NPs, and when the wavelength range was > 500 nm, there was no absorption observed (black line in Figure 2). Under the excitation of 385 nm (blue line in Figure 2), the maximum emission wavelength was 512 nm (red line in Figure 2). The aqueous solution of Mn-Si NPs was uniform and transparent light

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yellow liquid and showed bright green fluorescence under 365 nm UV lamp irradiation as shown in the inset in Figure 2. The fluorescence quantum yield of Mn-Si NP was 4.2% (Figure S5a) with quinine sulfate as a reference and the fluorescence lifetime value was 5.73 ns (Figure S5b, Table S1) which was longer than the autofluorescence of the living organism, whose lifetimes were shorter than 5 ns26. Figure S6a showed the fluorescence emission spectrum of Mn-Si NPs under different excitation wavelengths from 300 nm to 410 nm, which clearly illustrated excitation-independent emission of the as-prepared Mn-Si NPs.42 The as-prepared Mn-Si NPs possessed optical properties of a certain up-conversion as shown in Figure S6b. The emission wavelength was concentrated in 512 nm with the excitation wavelength changed from 600 to 850 nm. As shown in Figure S7, the as-prepared Mn-Si NPs had excellent chemical stability. PL intensity of Mn-Si NPs had very small changes in the pH range from 3 to 11, which demonstrated the excellent pH stability of Mn-Si NPs (Figure S7a). Mn-Si NPs had remarkable NaCl salt stability with the concentration of NaCl increasing from 5 to 100 mM (Figure S7b). As shown in Figure S7c, the PL of FITC dye was quickly quenched within 20 min irradiation under 488 nm excitation and only 60% left of its initial PL intensity after 60 min irradiation (black line in Figure S7c). Compared with FITC, the PL intensity of Mn-Si NPs decreased very little after 180 min irradiation under 385 nm excitation (green line in Figure S7c)，showing excellent photostability. We also found that the PL intensity of the as-prepared Mn-Si NPs was hardly reduced after 30 days, indicating the favorable storage stability of the asprepared Mn-Si NPs (Figure S7d). In vitro Cytotoxicity Assay and Imaging of Mn-Si NPs. MTT assay on A549 cells was used to evaluate the cytotoxicity of Mn-Si NPs in vitro. Roughly, the cell viability showed a dose-dependent manner at each time point. However, no statistical significance was found among groups at each time point. After the cells were incubated with 500 μg/mL of Mn-Si NPs for 24 h, the cell viability was slightly equal to 82% (Figure 3). This result demonstrated the low cytotoxicity of Mn-Si NPs in vitro. To further assess the cytotoxicity, we observed the morphology of A549 cells after 12 h of incubation with Mn-Si NPs. No obvious change was found in cell shape after being treated with Mn-Si NPs for 12 h at different concentrations (Figure 4). In cell imaging, no fluorescent signal was detected in the control group (Figure 4a). However, the cells in Mn-Si NPs-treated groups showed green fluorescence under 488 nm excitation, and the fluorescent signals increased in a dose-dependent pattern (Figure 4b-e). The merged pictures of Mn-Si NPs and the nucleus (Figure 4b’-e’) indicated that Mn-Si NPs successfully entered into the A549 cells and mainly located in the cytoplasm around the nucleus (Figure 4b’’-e’’). Combined with the result of cell viability, Mn-Si NPs are available for bioimaging in vitro. In order to explore the time lapse imaging, A549 cells were incubated with 500 μg/mL of Mn-Si NPs and photo-

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Figure 4. The confocal microscopic fluorescence pictures of A549 cells after incubating with RPMI-1640 medium containing 0, 150, 200, 300 and 500 μg/mL of Mn-Si NPs for 12 h. Green fluorescence photographs of Mn-Si NPs in the A549 cells (a-e), blue fluorescence photographs of the nucleus stained with DAPI (a’-e’), and merged images (a’’e’’). Scale bar: 20 μm.

Figure 6. Bright-field and fluorescence pictures of zebrafish embryos exposed to Mn-Si NPs. Notes: Brightfield photographs (a-e) and green fluorescence photographs (a’-e’) of embryos incubated with Mn-Si NPs at concentrations of 0 (a and a’), 25 (b and b’), 50 (c and c’), 100 (d and d’) and 200 (e and e’) μg/mL at 4 hpf. Scale bar: 300 μm.

Figure 7. Bright field and fluorescence pictures exposure to Mn-Si NPs on zebrafish embryos. Bright field images (a-e) and green fluorescence images (a’-e’) of embryos exposed to 50 μg/mL Mn-Si NPs at 12 (a and a’), 24 (b and b’), 36 (c and c’), 48 (d and d’), and 60 (e and e’) hpf. Scale bar: 300 μm.

Figure 5. The confocal microscopic fluorescence pictures of the A549 cells after incubating with 500 μg/mL Mn-Si NPs in RPMI-1640 medium at different time points (1 h, 3 h, 6 h and 12 h). Green fluorescence photographs of Mn-Si NPs (a-e), blue fluorescence photographs of nucleusstained with DAPI (a’-e’), and their merged images (a’’-e’’). Scale bar: 20 μm. graphed at different time points (1 h, 3 h, 6 h and 12 h). We found that the onset of Mn-Si NPs cellular uptake was detected after 1h, followed by an increase in the brightness up to 12 h (Figure 5). As the incubation time in creased, the emission brightness that was from Mn-Si NPs embedded in the cells was also intensified. The cells kept a decent morphology although the increased incubation time led to the slight-dehydration of the cells at 500 μg/mL. These results not only illustrated the low toxicity of Mn-Si NPs, but also demonstrated that Mn-Si NPs can be gradually taken up through endocytosis. Toxicity Test and Imaging of Mn-Si NPs on Zebrafish. The zebrafish is becoming an ideal model for assessing the bio-toxicity of nanomaterials and in vivo imaging in

Figure 8. (a) T1-weighted MR images of Mn-Si NPs with different concentrations of Mn2+. (b) The r1-relaxivity curve of Mn-Si NPs. recent years. Their contributed features include external development, transparent embryos at the early stage and the temporal-spacial pattern of development. 43,44 The size of Mn-Si NPs (ca.5.4 nm in diameter) was much smaller than chorionic aperture which is approximately 600 nm in diameter.45,46 Therefore, it is easy for Mn-Si NPs to go in the embryos via the chorionic pores of zebrafish. To assess


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Figure 9. In vivo T1-weighted MR images. MR images (a, b) of zebrafish embryos incubated with different concentrations of Mn2+ in Mn-Si NPs. (a) Sagittal plane. (b) Horizontal plane. (c) MR images of the mice before and after injection with Mn-Si NPs (25 μmol Mn2+ per kg). the toxicity of the Mn-Si NPs on zebrafish, survival rate, hatching rate and phenotype of the zebrafish embryos were monitored and followed throughout the embryonic development period (Figure S8 and S9).47 For the control group, the survival rate at 36 hpf and the hatching rate at 72 hpf were 85% and 82%, respectively. In the 50 μg/mL exposed group, these two values were 84% and 83%, respectively (Figure S8). No significant difference was found among groups either for the survival rate at 36 hpf (Figure S8a) or for the hatching rate at 72 hpf (Figure S8b). For phenotype, similar to the embryos in unexposed groups (Figure S9a-d), no obviously deformity was observed in exposed groups of embryos (Figure S9e-t) according to the standard at 4 hpf, 24 hpf, 48 hpf and 72 hpf. 48-50 The combining results of gross development with survival rate and hatching rate of the embryos demonstrated that an exposure to Mn-Si NPs had little effect on embryonic development. Figure 6 showed the fluorescence imaging of zebrafish embryos which were exposed with different doses of MnSi NPs. The fluorescent signals intensified in a dosedependent manner. Then we selected 50 μg/mL as the aqueous exposure dose to inspect the stability and the time lapse distribution of Mn-Si NPs in zebrafish embryos (Figure 7a-e’). The as-prepared Mn-Si NPs, as compared to be exposed for three hours (Figure 6c’), still showed green fluorescence at 12 hpf and mainly scattered in the yolk sac (Figure 7a’). Along with the development, the fluorescence intensity was progressively weakened and disappeared around 60 hpf (Figure 7b’-e’). It was previously reported that carbon dots were removed through the digestive system.47,51 We supposed that Mn-Si NPs might be eliminated via the same pathway. Meanwhile, at 60 hpf, the embryos showed a normal gross appearance (Figure 7e) that further indicated that the as-prepared Mn-Si NPs have low toxicity in vivo. MRI of Mn-Si NPs. In order to investigate the magnetic resonance imaging ability of the synthesized nanoparticles, we determined the different relaxation time (T1) of

Mn-Si NPs at different concentrations of Mn2+ (Figure 8a). The r1 relaxivity of Mn-Si NPs was 4.25 mM−1 s−1 (Figure 8b). As shown in Figure 8a, the magnetic resonance signal was strengthened with the increase of Mn2+ concentration. The above results indicated that the as-prepared Mn-Si NPs have potential as T1-weighted MRI agent. We further performed the in vivo T1-weighted MRI of zebrafish embryos52 and BALB/c nude mice on a 1.2 T MRI system (Figure 9). As shown in Figure 9a and Figure 9b, no magnetic resonance response was observed when zebrafish embryos were incubated with E3 medium, whereas the brightness of MRI was gradually brightened with increasing the concentration of Mn2+ in Mn-Si NPs. As shown in Figure 9c, compared with no injection, the liver and kidneys area of the mouse appeared obvious magnetic signals after 30 minutes of injection. The results indicated that the as-prepared Mn-Si NPs have the ability of magnetic

Figure 10. (a) H&E images of major organs (heart, liver, lung, spleen, and kidney) of mice (7 days) in control group (injected with normal saline) and treatment group ( injected with Mn-Si NPs). (b) The body weight changes of the mice injected with normal saline (n=3) and injected with Mn-Si NPs (n = 3).


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resonance imaging in vivo.

Notes

In vivo Toxicity of Mn-Si NPs in Mice. We performed histological examination and long-term body weight monitoring in mice to explore the toxicity of Mn-Si NPs in vivo (Figure 10). Compared with the control group, no tissue damage was found in mice injected with Mn-Si NPs after 7 days of culture (Figure 10a). We also assessed the longterm toxicity of Mn-Si NPs in vivo by regularly measuring body weight of the mice (Figure 10b). After 25 days of culture, there was no death in the control (injected with normal saline, n=3) and experimental (injected with MnSi NPs, n=3) groups, and the mice in control and experimental groups showed the same growth trend. The results indicated that the as-prepared Mn-Si NPs neither caused the damage to the tissues nor hindered the growth of mice.

The authors declare no competing financial interest.

CONCLUSION In summary, a method for synthesizing water-dispersible Mn2+ functionalized Si NPs (Mn-Si NPs) as fluorescence and MR dual-modality imaging probe under mild experimental conditions was reported. The whole synthesis process was carried out under room temperature and atmospheric pressure, and did not require any expensive andspecial equipment. The combination of Mn2+ and Si NPs not only retained the fluorescent property of Si NPs but also added the high spatial resolution advantages of magnetic resonance imaging. The as-prepared Mn-Si NPs showed fine pH stability, NaCl stability, photostability and high biocompatibility. The fluorescence imaging in A549 cell and zebrafish indicated that the as-prepared Mn-Si NPs could successfully enter into the biological body and showed low toxicity. At the same time, MRI in zebrafish embryos and mice testified that the as-prepared Mn-Si NPs had favorable magnetic resonance imaging ability in vivo. The combination of Mn2+ and Si NPs promoted the developments of further application of Si NPs in the field of biological imaging. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Chemicals and reagents, instruments, the optimization of synthetic reaction conditions, XPS spectra of Mn-Si NPs, the structure characterizations of Si NPs, the quantum yield, fluorescence lifetime, the optical properties of Mn-Si NPs aqueous solution, the chemical stability of Mn-Si NPs, cytotoxicity assay, bioimaging of Mn-Si NPs in vitro and in zebrafish, MRI of Mn-Si NPs in vitro and in vivo, toxicity of Mn-Si NPs in mice, and supplemental Table S1 and Figures S1−S9 (PDF).

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (nos. 21475069 and 81671179) and the Tianjin Natural Science Foundation (nos. 16JCZDJC37200 and 15JCYBJC24400). REFERENCES (1) Pathak, S.; Choi, S.-K.; Arnheim, N.; Thompson, M. E. J. Am. Chem. Soc. 2001, 123, 4103-4104. (2) Jaiswal, J. K.; Mattoussi. H.; Mauro, J. M.; Simon. S. M. Nat. Biotechnol. 2002, 21, 47−51. (3) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142-12150 (4) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science. 2003, 300, 1434-1436. (5) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11-18. (6) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stölzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331-338. (7) Das, P.; Saha, A.; Maity, A. R.; Ray, S. C.; Jana, N. R. Nanoscale. 2013, 5, 5732–5737. (8) Rosso-Vasic, M.; Spruijt, E.; Popovic, Z.; Overgaag, K.; Lagen, B. V.; Grandidier, B.; Vanmaekelbergh, D.; Domnguez-Gutierrez, D.; Cola, L. D.; Zuilhof, H. J. Mater. Chem. 2009, 19, 5926–5933. (9) Zou, Jing.; Baldwin, R. K.; Pettigrew, K. A.; Kauzlarich, S. M. Nano Lett. 2004, 4, 1181-11186. (10) Veinot, J. G. C. Chem. Commun. 2006, 4160–4168. (11) Heinrich, J. L.; Curtis, C. L.; Credo, G. M.; Sailor, M. J.; Kavanagh, K. L. Science. 1992, 255, 66−68. (12) He, Y.; Zhong, Y. L.; Peng, F.; Wei, X. P.; Su, Y. Y.; Lu, Y. M.; Su, S.; Gu, W.; Liao, L. S.; Lee, S.-T. J. Am. Chem. Soc. 2011, 133, 14192–14195. (13) He, Y; Kang., Z. H.; Li, Q. S.; Tsang, C. H. A.; Fan, C. H.; Lee, S.-T. Angew. Chem., Int. Ed. 2009, 48, 128−132. (14) Kang, Z. H.; Tsang, C. H. A.; Zhang, Z. D.; Zhang, M. L.; Wong, N.-B.; Zapien, J. A.; Shan, Y. Y.; Lee, S.-T. J. Am. Chem. Soc. 2007, 129, 5326-5327. (15) Kang, Z. H.; Liu, Y; Tsang., C. H. A.; Ma, D. D. D.; Fan, X.; Wong, N.-B.; Lee. S.-T. Adv. Mater. 2009, 21, 661−664. (16) Wu, M. H.; Mu, R.; Ueda, A.; Henderson, D. O.; Vlahovic, B. Mater. Sci. Eng. B. 2005, 116, 273−277. (17) Erogbogbo, F.; Yong, K.-T.; Roy, I.; Xu, G. X.; Prasad, P. N.; Swihart, M. T. ACS Nano. 2008, 2, 873−878.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Fax: +86-22-23502458. *E-mail: [email protected]. Fax: +86-22-23502554.

(18) He, G. S.; Zheng, Q. D.; Yong, K.-T.; Erogbogbo, F. M.; Swihart, T.; Prasad, P. N. Nano Lett. 2008, 8, 2688−2692.


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Synthesis of Water-Dispersible Mn2+ Functionalized Silicon

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