Supporting Information. Shape-controlled Synthesis of Colloidal Superparticles from Nanocubes

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1 Supporting Information Shape-controlled Synthesis of Colloidal Superparticles from Nanocubes Tie Wang 1, Xirui Wang 1, Derek LaMontagne 1, Zhongliang Wang 1, Zhongwu Wang 2, and Y. Charles Cao 1* 1 Department of Chemistry, University of Florida, Gainesville, FL 32611, United States. 2 Cornell High Energy Synchrotron Source, Wilson Laboratory, Cornell University, Ithaca, New York 14853, United States. * To whom correspondence should be addressed: cao@chem.ufl.edu (Y.C.C.) This PDF file includes: Materials and Methods Figures S1-S8 Table S1 References S1

2 1. Chemical and biological reagents. Iron(III) chloride hexahydrate (FeCl 3 6H 2 O 98%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), polyvinylpyrrolidone (PVP, MW=55,000), fetal bovine serum (FBS), streptomycin, penicillin, and ethylene glycol (EG, 99%) were purchased from Aldrich. Dodecyltrimethylammonium bromide (DTAB, 97%) was purchased from Alfa Aesar. Sodium oleate (95%) was purchased from technical chemical laboratories (TCL). The LIVE/DEAD cell viability kits were purchased from Invitrogen (Carlsbad, CA, USA). The CellTiter-Blue cell viability kits were purchased from Promega (San Luis Obispo, CA, USA). All other chemicals, biological reagents, and solvents were purchased from Fisher Scientific International, Inc. Nanopure water was made by a Barnstead Nanopure Diamond system. 2. Calculation of surface area and volume for the Fe 3 O 4 superparticles. The surface area S and volume V of cubic Fe 3 O 4 superparticles (Figure S1A) is given by: S 6 a V a The surface area S and volume V of smooth spherical Fe 3 O 4 superparticles (Figure S1B) is found by: S 4 π R V β 4 3 π R The surface area S and volume V of rough spherical Fe 3 O 4 superparticles (Figure S1C) is determined by the following equations: S σ 6 π R V β 4 3 π R where σ π R S π R 1 S π R 1 4 πr 1 cosθ S2

3 β 4 3 π R V π R V π R 2 1 cosθ πr S and V are the surface area and volume of the void part in the circular base (Figure S1D). When l is 14 nm, R is equal to 130 nm, and σ and β are estimated to be and 0.998, respectively. So and S R nm m S R nm m When V V, the length (a) of cubic superparticles is 1.61 times larger than the radius R of spherical superparticles, which means a 1.61 R 209 nm. Here, the volume of cubic or rough spherical superparticles is and the surface area is V V nm m, S R nm m 3. Synthesis of 11.0-nm Fe 3 O 4 nanocubes. The nanoparticles were prepared by thermal decomposition of iron(iii) oleate by modifying previous literature methods. 1 Briefly, sodium oleate (48.7 g, 160 mmol) and FeCl 3 6H 2 O (10.8 g, 40 mmol) were dissolved in n-hexane (140 ml), ethanol (80 ml), and deionized H 2 O (60 ml). The mixture was refluxed for 4 h, the organic phase was separated and washed with nanopure H 2 O, and the solvent was removed on a rotary evaporator. Then, iron oleate (0.9 g) and OA (0.14 g) were dissolved in ODE (5 ml) and the solution was heated to 320 C. After refluxing for 30 minutes, the synthesis solution was cooled to room temperature, and the resulting Fe 3 O 4 particles were purified by using the solvent/non-solvent pair n-hexane/ethanol four S3

4 times. TEM observations show that the resulting particles appear as nanocubes having 11.0 nm in length with a standard deviation of 4.8% (Figure S2). 4. Thermogravimetric analysis (TGA). Thermogravimetric analysis (TGA) was used to detect the amount of OA on the Fe 3 O 4 nanocubes. After purification, the sample was dried in a vacuum oven overnight at 50 C with a reduced pressure (30 in. Hg), and then the resulting sample was scanned from 25 C to 700 C under TGA (Figure S3). The mass percent of ligand molecules on the Fe 3 O 4 nanocubes was 9.3%. Therefore, on average, there are 1483 oleate ligands on each Fe 3 O 4 nanocubes. This number corresponds to a 40.8% surface coverage if one assumes an average coverage area of an oleate ligand on the Fe 3 O 4 nanocubes to be 0.2 nm Synthesis of sphere-shaped superparticles from Fe 3 O 4 nanocubes. 2 Typically, a chloroform solution of 11.0-nm Fe 3 O 4 nanocubes (28 µm, 1.0mL) was thoroughly mixed with a DTAB (20.0 mg, 65.0 µmol) solution by a vortex mixer. Afterwards, the chloroform was removed from the mixture by bubbling Ar at 40 C. A clear, dark nanoparticle-micelle aqueous solution was obtained. This Fe 3 O 4 nanocube-micelle aqueous solution was injected into a three-neck flask with a PVP ethylene glycol solution (2.0 mm, 5.0 ml) under vigorous stirring for 10 min. The resulting colloidal superparticles were separated by centrifuge (3,300 rpm, 5 min). The black precipitate was redispersed into ethanol and the superparticles were further purified twice by centrifugation. Spherical superparticles with a diameter of 260 nm and a standard deviation of 14% were made (Figure 2A). 6. Synthesis of cube-shaped superparticles from Fe 3 O 4 nanocubes. 2 In a typical synthesis, a chloroform solution of 11.0-nm Fe 3 O 4 nanocubes (28 µm, 1.0mL) was thoroughly mixed with a solution containing DTAB (20.0 mg, 65.0 µmol) and oleic acid (0.04 ml) by a vortex mixer. Afterwards, the chloroform was removed from the mixture by bubbling Ar at 40 C. A clear, dark nanoparticle-micelle aqueous solution was obtained. This Fe 3 O 4 nanocube-micelle aqueous solution was injected into a three-neck flask with an ethylene glycol solution containing (2.0 mm, 5.0 ml) under vigorous stirring for 10 min. The resulting colloidal superparticles were separated by centrifuge (3,300 rpm, 5 min). The black precipitate was redispersed into ethanol and the S4

5 superparticles were further purified twice by centrifugation. TEM observations show that the resulting superparticles exhibit a cubic shape with an edge length of 209 nm and a standard deviation of 18% (Figure 2C). However, if more than 0.10 ml of OA was added during the synthesis, aggregated cubic superparticles were observed instead of dispersed cubic superparticles (Figure S4). 7. Small-angle XRD measurements. Small-angle X-ray diffraction spectra were measured inside a diamond anvil cell (DAC) where two well-aligned diamond anvils have a culet size of 0.5 mm. Sphere- and cube-shaped superparticles of ~1 mg were used in a typical measurement, and the measurements were performed at room temperature using an angle dispersive synchrotron X-ray source at the B 2 station of Cornell High Energy Synchrotron Source (CHESS). A double-bouncing Ge (111) monochromator converts incident white X-rays to a monochromatic beam. The sphere-shaped superparticles were measured using a X-ray source with an energy of 17.0 kev (the corresponding wavelength is 0.727,692 Å), whereas the cube-shaped superparticles were measured using an X-ray source with an energy of 18.0 kev (the corresponding wavelength is 0.688,889 Å). The lattice constant (a) was calculated by the d spacing between superlattice planes with Miller indices {hkl} using the formula: a d h k l. 8. Cellular uptake. In a typical experiment, human LNCaP prostate tumor cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 µg ml -1 streptomycin and 100 U ml -1 penicillin) in a humidified atmosphere at 37 o C with 5% CO 2 (Sanyo CO 2 incubator, type MCO-17AIC). After seeding LNCaP cells in a 24-well plate for 24 hours, the Fe 3 O 4 superparticles in water (100 µl, 0.5 nm) were added to the cell culture and incubated for 12 hours. Afterwards, the culture media was removed. The cells were washed with phosphate buffered saline three times, and detached from the six-well plates using trypsin (1.0 ml, 0.25%) to transfer into glass vials. The number of cells in each well was counted using a hemocytometer (~ cells/well). To compare the cellular uptake of spherical and cubic superparticles, the cells were lysed under sonication (Branson 2510, 37 C) for 1 h, and clear solutions were obtained. The Fe 3 O 4 S5

6 superparticles in the solution were dissolved using 20% HNO 3 (0.2 ml), and then diluted by 10. The concentrations of iron ions were determined via an inductively coupled plasma mass spectrometer (ICP-MS element 2 ICP-MS, Thermo Scientific, USA). Finally, the concentration of Fe 3 O 4 superparticles in each well was calculated by the iron ion concentration and the size of the superparticles determined by TEM. Finally, the number of Fe 3 O 4 superparticles per cell in each well was calculated using the number of cells in the corresponding well (cubic superparticles: 268 ± 24 / cell; spherical superparticles: 271 ± 20 / cell) Magnetic field induced cell death (LIVE/DEAD cell viability). In a typical experiment, human LNCaP prostate tumor cells were cultured in RPMI-1640 medium, supplemented with 10% FBS and antibiotics (100 µg ml -1 streptomycin and 100 U ml -1 penicillin) in a humidified atmosphere at 37 o C with 5% CO 2 (Sanyo CO 2 incubator, type MCO-17AIC). After seeding LNCaP cells in a 24-well plate for 24 hours, the Fe 3 O 4 superparticles in water (100 µl, 0.5 nm) were added to the cell culture and incubated for 12 hours. The cells were then thoroughly washed and placed in fresh culture media. Afterwards, the plates with the cells were placed in a home-made magnetic stimulator which consists of two pairs of magnets (Figure 3A). The size of the magnets is cm, and the distance between the magnet pairs is 2 cm. The strength of the magnetic field in the center is 4.5 kg. This magnetic stimulator leads to a 4-Hz pulsed magnetic field with a gradient ( B) of 64.8 kg/cm. After 10 minutes of treatment, the magnetic field was removed. The cells were recovered in a humidified atmosphere at 37 o C with 5% CO 2 for 2 hours, and then the cells were stained with a LIVE/DEAD cell viability kit (Invitrogen, Carlsbad, CA) following the manufacturer s manual. Live cells with green fluorescence (494 Ex/517 Em nm) and dead cells with red fluorescence (517 Ex /617 Em nm) were imaged using a fluorescence microscope (TE-2000 Nikon EZ-C1 confocal microscope) under 10x magnification Quantitative evaluation of cell death (CellTiter-Blue cell viability assay). In a typical experiment, human LNCaP prostate tumor cells were cultured in RPMI-1640 medium, supplemented with 10% FBS and antibiotics (100 µg ml -1 streptomycin and 100 U ml -1 penicillin) in a humidified atmosphere at 37 o C with 5% CO 2 (Sanyo CO 2 S6

7 incubator, type MCO-17AIC). LNCaP cells were seeded in a 96-well plate ( cells per well) for 24 h, and then an aqueous solution of sphere- or cube-shaped Fe 3 O 4 superparticles (100 µl, 0.5 nm) were added to the cell culture and incubated for 12 h. After washing and changing with fresh culture media, the cells were introduced into a 4- Hz pulsed magnetic field, and then were recovered in a humidified atmosphere at 37 o C with 5% CO 2 for an additional 2 h. Then CellTiter-Blue cell viability assay (Promega, San Luis Obispo, CA) was used to quantify the dead cells versus live cells after this treatment. After adding CellTiter-Blue Reagent (20 µl) to each well and incubating for 4 h, fluorescence at 560 Ex /590 Em nm was recorded using a 96-well plate reader (Infinite M200 PRO series from Tecan Group Ltd). 11. Forces induced by a rotating magnet. 4-6 The magnetic strength around a magnet is non-uniform. relationship The magnetic induction B is related to the magnetization M by the M χ μ B where χ χ χ is the difference in magnetic susceptibilities (dimensionless) between particle χ and the surrounding medium χ, μ 4π 10 T m A is the permeability of vacuum, and V is the volume of the Fe 3 O 4 superparticles (m 3 ). As the value of χ for human tissue is very small, χ is negligible for this calculation. A sphere-shaped Fe 3 O 4 superparticle of diameter D nm has a nearly identical volume to that of a cube-shaped Fe 3 O 4 superparticle (length of nm). Considering the ligands on the surface, the total mass of each single superparticle is g. An induced magnetic field B = 4.5 kg is applied to the Fe 3 O 4 superparticles. Because of the non-uniformity of magnetic strength, there is the existence of a magnetic gradient ( B kg/m ) around the magnet, and the force induced by this external magnetic field varies in different positions. The moment (m) of a single cubic or spherical Fe 3 O 4 superparticle equals emu. For such large magnetic fields, the force is proportional to the gradient of the magnetic field: 7 S7

8 m B emu kg m femtonewton fn Figure S1. Models of Fe 3 O 4 superparticles. (A) Cubic shape, (B) the spherical shape with a smooth surface, (C) the spherical shape with a rough surface, and (D) nanocube S8

9 packing on the top of a circular base with a radius of R: the length of the cube (a), the angle θ = 45o, and the length of cubic nanoparticles (l). Figure S2. TEM images of Fe3O4 nanocubes under different magnification. S9

10 Figure S3. TGA data of Fe 3 O 4 nanocubes bonded by OA ligands. Figure S4. TEM images of the aggregated superparticles formed by adding OA (0.10 ml) into Fe 3 O 4 nanocubes solution (28 µm, 1.0mL) S10

11 Table S1. From these data, the value for the lattice constant of spherical and cubic superparticles were found to be 14.0 ± 0.2 nm and 14.1 ± 0.2 nm, respectively. Spherical Superparticles Cubic Superparticles hkl d(nm) a(nm) hkl d(nm) a(nm) * * * * * The d spacings of the (220) and (300) peaks were determined from zoomed-in spectra. S11

12 Figure S5. Magnetic response of Fe 3 O 4 superparticles over a wide field ranging from -5 koe to 5 koe with a SQUID magnetometer at room temperature (300 K). The magnetization of the Fe 3 O 4 superparticles saturates at 70.4 (emu/g) in fields stronger than 1 koe. S12

13 Figure S6. Fluorescent images of cancer cells loaded by Fe 3 O 4 superparticles: (A) and (B) spherical superparticles, (C) and (D) cubic superparticles. Green color represents the living cells and red color indicates the dead cells. S13

14 Figure S7. Schematic illustration of the collision of the lipid bilayer membrane with (A) sphere-shaped Fe 3 O 4 superparticles, and (B) cube-shaped Fe 3 O 4 superparticles. Figure S8. Schematic illustration showing how the shape of the superparticles influences the integrity of the cell membrane: (A) the membrane embedded by a spherical Fe 3 O 4 superparticle has a small void, while (B) the membrane has larger leaking voids when a cubic Fe 3 O 4 superparticle penetrates in. S14

15 Supplementary References 1. Park, J. et al, Nature Mater. 2004, 3, Wu, H.; Zhu, H.; Zhuang, J.; Yang, S.; Liu, C.; Cao, Y. C. Angew. Chem. Int. Ed. 2008, 47, Wang, Z.; Liu, H.; Yang, S. H.; Wang, T.; Liu, C.; Cao, Y. C. Proc. Nat. Acad. Sci. 2012, online. 4. Shevkoplyas, S. S.; Siegel, A. C.; Westervelt, R. M.; Prentiss, M. G.; Whitesides, G. M. Lab Chip, 2007, 7, Dobson, J.; Cartmell, S. H.; Keramane, A.; Haj, A. J. E.; IEEE T Nanotechnol. 2006, 5, Grumann, M.; Geipel, A.; Riegger, L.; Zengerle, R.; Ducrée, J. Lab Chip, 2005, 7, Lipfert, J.; Hao, X.; Dekker, N. H. Biophys. J. 2009, 96, S15