Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006
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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006
2 Supporting Information Specific targeting, cell sorting and bio-imaging with smart magnetic core-silica shell nanomaterials Tae-Jong Yoon, 1+ Kyeong Nam Yu, 2+ Eunha Kim, 1 Jun Sung Kim, 2 Byung Geol Kim, 1 Sang-Hyun Yun, 3 Byeong-Hyeok Sohn, 1 Myung-Haing Cho, 2 Jin-Kyu Lee, 1 * Seung Bum Park 1 * 1. School of Chemistry, Seoul National University, Seoul, , Korea; 2. College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, , Korea; 3. Department of Materials Science and Engineering, Pohang University of Science and Technology, Pohang, , Korea Standard Fmoc Quantification Protocols: Fmoc protected MNP@SiO 2 (OD)-PEG/NH 2 s were precisely weighed about 2 mg in an Eppendorf tube through the separation by centrifugation followed by drying under high vacuum overnight. Precipitated MNPs were resuspended with 0.8 ml of DMF by sonication for 20 minute, followed by addition of 0.2 ml of piperidine for Fmoc cleavage. The cleavage cocktail with MNPs was sonicated for 20 minutes. The supernatant was collected by centrifugation at 13,000 rpm for 20 minutes, and the UV absorbance of Fmoc solution (0.9 ml in cell) was obtained at 300 nm is the extinction coefficient (units mol -1 dm 3 cm -1 ) at 300 nm. For greater accuracy, each sample was tested in heptaplicate and UV absorbance should lie between about 0.3 and 1.2 absorbance units. 1
3 Cytotoxicity of superparamagnetic Silica coated MNPs vs. Quantum Dots: Cytotoxicity of quantum dots as like Cadmium chalcogenide (CdSe) has been reported recently in in vitro cells (Nano Lett. 2005, 5, and Angew. Chem. Int. Ed. 2005, 44, ). A Key point of above-mentioned paper from referee 1 is the introduction of silica shell to eliminate cytotoxicity from cadmium ion bleaching and the preparation of magnetic and fluorescent quantum dot hybrid system for the application in in vitro and in vivo bio-imaging. However, they were failed to enumerate the real application toward biosystem, and simply proposed the applicability in bio-imaging, which clearly has great potentials. In our report, fluorescence organic dyes with high quantum yields were utilized as one of the detection methods as quantum dot (Nano Lett, 2005, 5, ), and the photobleaching, an inherent problem of fluorescence organic dyes was resolved by embedding them in the silica-shell (see the manuscript for detail). We performed making of silica shell, fabrication of silica surface with multifunctional chemicals and quantitative attaching of bio-molecules such as sequence-independent immobilization of antibodies for specific targeting. Thus, we successfully enumerate the bioapplication in illustration of our smart silica shell-magnetic core nanoparticles (in vivo and in vitro). 2
4 @ Figure 1. Powder X-ray Diffraction pattern of 2 (FITC) X-ray powder diffractogram shows the typical diffraction pattern of spinel structure, confirming the core structure of magnetic Co-ferrite nanoparticle. The data was collected using Philips PW 3710 powder diffractometer equipped with Ni-filtered Cu K-radiation (λ = Å). Then, according to the Scherrer equation, the roughly spherical particles size is related to the width of the diffracted beams. Using the full width at half-maximum of the most intense X-ray peak, which corresponds to the [311] one, it gives in thecase of our samples the nanoparticles mean diameter of 9 nm. Co ferrite Cubic cells deduced from the analysis of x-ray diffraction patterns (lattice parameter, a exp = 0.835). Increasing of peak broadness at the low angle was occurred from amorphous silica shell. 3
5 @ Figure 2. Dynamic light scattering (DLS) data of the 2 (FITC) magnetic core-silica shell nanoparticle in H 2 O. The correlation graph and size distribution graph are not shown, and below graph is calculated from the light scattering theorem. The size of core-shell nanoparticles is exactly determined 58.1 nm as shown in the DLS size distribution graph. Dynamic light scattering measurements were performed using a UNIPHASE He-Ne laser operating at nm. The maximum operating power of the laser was 30 mw. The detector optics employed optical fibers coupled to an ALV/SO-SIPD/DUAL detection unit, which employed an EMI PM-28B power supply and ALV/PM-PD preamplifier/discriminator. The signal analyzer was an ALV- 5000/E/WIN multiple tau digital correlator with 288 exponentially spaced channels. Its minimum real sampling time is 10-6 s and a maximum of about 100 s. A lens with a focal length of 200 mm narrowed the incident beam to reduce the thermal lensing effect and to increase the coherence area. The scattered beam passed through two pin holes before reaching the PMT. A scattering cell (10 mm diameter cylindrical) was placed in a temperature controlled bath of index matching liquid, decaline. All of the experiments were performed at 25 ± Figure 3. Vibrato Sample Magnetometer (VSM) data of MNP@SiO 2 (FITC) magnetic core-silica shell nanoparticles Magnetic properties of core-shell nanoparticles were measured by Vibrating Sample Magnetometer (VSM, Lake Shore Model 7304). The nanoparticles were already described in Fig. 1 of the main text. The coercivity (H c ) value was exactly same for bare Co ferrite nanoparticles. 4
6 @ Figure 4. MTT assay data of 2 (FITC) for MCF-7 cell (black), A549 (light gray), and NL20 (gray). In this assay, the cell viability was maintained at greater than 85 % in all groups indicating that the core-shell nanoparticle do not show acute cytotoxicity to various cells at the level of a few tenths of micrograms (80 µg ml -1 ) within 48 h. The cells were incubated in a 96-well plate. At the end of the incubation period, 3(4,5-dimethylthiazol- 2-yl) 2,5-diphenyltetrazolium bromide (MTT; 50 µl, Sigma-Aldrich) in PBS (0.2 mg ml -1 ) was added to each well (final concentration of 0.4 mg ml -1 ) and cultures were incubated in 5 % CO 2 for 4 h at 37 C. Then the culture medium was carefully removed by pipetting and formazan crystals generated by dehydronase activity in mitochondria, which only occurs in living cells, were dissolved in DMSO for the analysis. After 10 min agitation on a shaker, absorbance was measured at 490 nm and 620 nm for test and reference solutions, Figure 4. TEM images of MNP@SiO 2 (RITC) uptakes cell. B is enlarged from black dot square in A. the core-shell structure of nanoparticles were clearly shown at the cytoplasm of the cell. 5
7 @ Figure 5. Bioapplication of smart 2 toward MRI imaging Maghemite nanoparticle, which is called superparamagnetic nanoparticles, has been studied in MRI. Our cobalt ferrite magnetite nanoparticle is also detectable with commercial MRI equipment. Actually a magnetite (MFe 2 O 4, M = Fe, Co, Mn.) nanoparticles have higher magnetization value (Ms) than a maghemite (Fe 2 O 3 ) nanoparticles (R. M. Cornell, U. Schwertmann, The Iron Oxides VCH, 1996, p 117). And CoFe 2 O 4 is a little higher Ms than Fe 3 O 4 (Langmuir, 2005, 21, ). To support our claim for the applicability of our system toward MRI imaging, the results of MRI study with our system were enclosed in the supporting information. The complete long-term distribution study is currently underway. The MRI signal of our smart MNP@SiO 2 system was provided as negative sign (black signal) as shown in Figure 1B and 1C of the main text. Our nanoparticles were successfully detected in in vivo mouse model by commercial MRI instrument (4.7 Tesla). According to this study, the unmodified MNP@SiO 2 was accumulated in the liver when it was administered through IP, which is clearly enumerated in a series of figures Figure 6. Video file of targeted floating SP2/0 cells moving by external magnet. Please open attached file (file name: sbpark(supplementary_video).avi). 6
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