Supporting Information

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1 Supporting Information Dumbbell-Like Au-Fe 3 Nanoparticles for Target-Specific Platin Delivery Chenjie Xu, Baodui Wang, and Shouheng Sun* Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA Experimental: Materials and Instrumentation: All chemicals including α,ω-bis{2-[(3-carboxy-1- oxopropyl)amino]ethyl}polyethylene glycol (M r = 3000) and (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) (MTT) were purchased from Sigma Aldrich Corp. RIPA buffer (25mM Tris HCl ph=7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) was mixed with Halt TM Protease inhibitor Cocktail before use (Pierce Corp.). Deionized (DI) water was purified by a Millipore Milli-DI Water Purification system. UV/Vis absorption spectra of the samples were measured with a PerkinElmer Lambda 35 UV/Vis spectrometer. Transmission electron microscopy (TEM) images were acquired with Philips EM 420 (120kV) on amorphous carbon coated copper grids. Cisplatin-binding Ligand (L2H) Synthesis: To a solution of cystamine dihydrochloride (1.125 g, 5 mmol) in 100 ml acetone and 10 ml Et 3 N were added ethyl bromoacetate (2.2 ml, 20 ml), KI (520 mg). After stirred for 6 h at room temperature, the insoluble solid was removed by filtration. The filtrate was dried in a rotavapor. The intermediate product was purified through flash chromatography (petrol/etoac, 20:1). Yield: 80%. FAB-MS: m/z = 519[M+Na] +. 1 H NMR (CDCl 3, 300MHz): d (8H, m, 4-H), 3.51 (8H, s, 5-H), (4H, q, 2-H), (4H, q, 3-H), (12H, t, 1-H). The deprotection of carboxylic group was carried in the methanol solution (0.496 g, 1 mmol). With 5 ml of 1 M NaOH aqueous solution, the mixture was stirred for 30 min. If there was any precipitate, small amount of water was needed. After 24 h of stirring, 20 ml distilled water was added and the solution was acidified to ph = 3.0 with 1 M HCl (aq). The resulting precipitate was collected by centrifugation and washed with EtOH/H 2 O (1:1). FAB-MS: m/z = 385[M+H] +. 1 H NMR (D 2 O, 300MHz): d 3.51 (8H, s, 3-H), (4H, t, 2-H), (4H, t, 1-H). Current address: College of Chemistry and Chemical Engineering and State S1

2 Synthesis and Modification of Dumbbell Nanoparticles: Au-Fe 3 nanoparticles were synthesized and made water-soluble according to our previous work. [1,2] To link Her2 antibody (Herceptin) to nanoparticles, nanoparticles (5 mg) in water was mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.1 mmol) and sulfo-nhs (1 mmol) for 15 min. Then the conjugates run through PD-10 column pre-washed with PBS to remove excessive EDC and sulfo-nhs. Then Herceptin (100 µg) was added into the PBS solution and shaked for 2 h. The final conjugates were separated from unbound Herceptin with high speed centrifugation. Linking Cisplatin onto Dumbbell Nanoparticles: The antibody-coupled Au-Fe 3 nanoparticles (1 mg) were mixed with cisplatin binding ligand solution (10 mmol, 1 ml H 2 O) for 6 h. Later, the uncoupled ligand was removed through PD-10 column or stirred cell (large amount synthesis). Cisplatin suspension (water, 20 mg/ml) was added to the nanoparticles solution. After stirred for overnight in dark, free cisplatin was separated from nanoparticles through low speed centrifugation (3000 rpm). The NPs then run through PD-10 column to remove free cisplatin in solution. The amount of platin was determined by ICP-AES. Cisplatin Release: Herceptin-Fe 3 -Au-platin NPs with 100 Pt µg/2 ml were put into dialysis bag (MWCO=1000, Spectrum Lab Corp), which was in 30 ml PBS bath at 37 o C. At certain time point, 1 ml PBS was sampled. The platinum concentration was determined by ICP-AES. Cell Experiments: Sk-Br3 cells were purchased from ATCC and cultured in a glass-bottom Petri dish (MatTek Corp.) with Dulbecco s Modified Eagle s Medium (DMEM) with 10% FBS and 1% antibiotics. Before incubation with nanoparticle conjugates, the cells were washed with PBS two times and blocked with 1% bovine serum albumin (BSA) in PBS. The nanoparticles solution in DMEM was incubated with cells for 1 h. Then, those cells were washed with PBS three times and fixed by 4% paraformaldehyde solution. After 30 min, the cells were washed with PBS for reflection imaging using a Leica TCS SP2 AOBS spectral confocal microscope. Cell Viability Test. Viability of Sk-Br3 cells incubated with Fe 3 -Au-platin, Herceptin-Fe 3 -Auplatin or cisplatin were examined through MTT assay. This cell viability test was based on the reduction of the tetrazolium salt MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by mitochondrial reductase in metabolically active cells. The cells were seeded onto 96-well culture plates at a density of 4000 cells per well in DMEM (200 µl) containing 10% FBS. After 24 h incubation at 37 o C, nanoparticles in DMEM buffer at different concentrations were added. The particles were removed after 24 h incubation. Then MTT solution (5 mg/ml in PBS) was added to each well to evaluate cell viability. After 1 h at 37 o C, the solution was removed. 100 µl DMSO was added to dissolve cells. Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou , P.R. China S2

3 After 30 min incubation at 37 o C, the viability was measured by microreader. Preparation of Sk-Br3 cell sample for TEM: Nanoparticles were dispersed in the cell culture medium (DMEM with 10% FBS, 1% penicillin) at a concentration of 0.01 mg Fe/mL dispersion. The mixture was incubated for 2 h and washed twice with PBS to remove the excess particles. The cells were detached with 0.05% trypsin EDTA and fixed with modified Karnovsky s Fixative (2% paraformaldehyde and 2% gluteraldehyde in PBS) before they were post-fixed in 1% Os for 1.5 h, stained with 2% uranyl acetate for 2 h, and dehydrated in alcohol and propylene oxide. The treated cells were then embedded in Eponate resin, sectioned with an ultramicrotome, and mounted on the 150 mesh TEM grids. The sections were then stained again with uranyl acetate (25 min) and lead citrate (10 min) for TEM image analysis. The images were acquired from a Philips EM 420 at 80 kv. p53 Protein Detection with Western Blot: 100,000 Sk-Br3 cells were plated in each well of a 6 well plate for 24 h. Au-Fe 3, platin-au-fe 3, platin-au-fe 3 -Herceptin or cisplatin in 1mL DMEM were added. After 16 h incubation, cells were collected and washed with cold PBS twice. Then 200 µl cold RIPA buffer was added to the cells and kept in ice for 40 min. The cell lysate was gathered through centrifugation at 14,000 rpm for 15 min. The concentration of protein for each sample was measured through Bradford protein assay. [3] 20 µg protein was loaded in each well for SDS-PAGE. The proteins are transferred to nitrocellulose membrane and blotted with Santa Cruz monoclonal p53 antibody (DO- 1). Calculation of Pt number per Au NPs: Each Au NP has fcc structure with a=0.408nm. [1] Each periodic lattice has 4 atoms. Atomic radius of Au atom is nm. Each Au NP is a sphere with diameter 8nm. Volume of Au NPs: m 3 Volume of lattice is m 3 Thus the number of atom of per NPs is Weight percentage of Pt/Au is 17.8%, thus atom ratio of Pt/Au is 17.9:100. Take into the number of gold atom per NPs, Pt/AuNPs is 2812 S3

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5 Figure S3. EDS characterization of S to Pt ratio for platin-au-fe 3 -Herceptin NPs. Au-Fe 3 NPs With Ligand (molar ratio) Without Ligand (molar ratio) Au/Fe Pt/Fe Pt/Au Pt/Fe Pt/Au 3-18 nm nm nm nm Table S1. ICP-AES analytical results in Au-Fe 3 NPs for platin loading with or without platin binding ligand. Percentage (%) Au-Fe 3 in Hexane Au-Fe 3 in Water Au-Fe 3 -Ab platin-au-fe 3 -Ab Size (nm) Figure S4. Hydrodynamic diameter of Au-Fe 3 nanoparticles at various functionalization stages. S5

6 Figure S5. TEM image of the platin-au-fe 3 -Heceptin nanoparticles in Sk-Br3 cells after two hour incubation. Figure S6. (A) Viability of Sk-Br3 cells after incubation with Au-Fe 3, Fe 3 -Au-platin and Herceptin-Fe 3 -Au-platin NPs under the same iron concentration; (B) Viability of MCF7 cells after incubation with Fe 3 -Au-platin, Herceptin-Fe 3 -Au-platin and cisplatin under the same platinum concentration; (C) p53 expression in Sk-Br3 cells after incubation with different concentrations of cisplatin; (D) p53 expression in Sk-Br3 cells after incubation with Au-Fe 3, Fe 3 -Au-platin, Herceptin-Fe 3 -Au-platin or cisplatin under the same Pt concentration (1 µg/ml) or Fe concentration (45 µg/ml). References: (1) Yu, H.; Chen, M.; Rice, P. M.; Wang, S. X.; White, R. L.; Sun, S. H. Nano Lett. 2005, 5, (2) Xu, C. J.; Xie, J.; Ho, D.; Wang, C.; Kohler, N.; Walsh, E.; Morgan, J.; Chin, Y. E.; Sun, S. H. Angew. Chem. Int. Ed., 2008, 47, (3) Sapan, C. V.; Lundblad, R. L.; Price, N. C. Biotechnol. Appl. Biochem. 1999, 29, S6