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1 Nanowired three dimensional cardiac patches Tal Dvir, Brian P. Timko, Mark D. Brigham, Shreesh R. Naik, Sandeep S Karajanagi, Oren Levy, Hongwei Jin, Kevin K. Parker, Robert Langer and Daniel S. Kohane NATURE NANOTECHNOLOGY 1

2 Figure S1 Nanowire statistics. Histogram of the length distribution of a typical sample of NWs. Figure S2 Photographs of scaffolds. a, Pristine alginate scaffold. b, NW-containing alginate scaffold (Alg-NW). The scaffolds were 5 mm in diameter and 2 mm high. 2 NATURE NANOTECHNOLOGY

3 SUPPLEMENTARY INFORMATION Figure S3 Mechanical properties of the Alg-NW. a, The Alg-NW prior to lyophilization. b, At higher concentrations of NWs the biomaterial became more viscous. c, Increasing NW concentration increased the elastic shear modulus of the biomaterial. N= 3 for each testing. Figure S4 NWs (0.5 mg/ml) embedded within the scaffold pore wall. a, NWs (yellow arrows) parallel to the scaffold pore wall. b, Elemental analysis of the features within the pore wall indicates the presence of gold. NATURE NANOTECHNOLOGY 3

4 Figure S5 Effect of increased loading of gold nanowires on the conductivity of alginate film. a, Topography of NWs (2 mg/ml) embedded in alginate film. b, Conductivity through the same region of nanowire-containing film as measured by C- AFM. 4 NATURE NANOTECHNOLOGY

5 SUPPLEMENTARY INFORMATION Figure S6 Effect of gold nanrods on the conductivity of alginate film. a, Gold nanorods by TEM. The rods were nm in diameter with a length scale of 60 nm. b, Topography of gold nanorods embedded within an alginate film by conductive-probe AFM (C-AFM). The field of view is 5 x 5 µm. c, Conductivity through the same region of nanorod-containing film as measured by C-AFM. d, The current vs. bias voltage over the range of -1 to 1V was negligible when the C-AFM tip was positioned over either a rod (red trace) or alginate (blue trace). NATURE NANOTECHNOLOGY 5

6 Figure S7 Cytotoxicity assay. Cell viability within the scaffolds was assessed by a metabolic activity assay (XTT assay). Results are normalized to day 0 values. N= 6 for each group at each time point. Figure S8 Protein expression. Troponin I (red) and Connexin 43 (green) co-staining of 3D cardiac constructs on day 3 for a, cardiac cells within pristine alginate scaffolds and b, cells within the Alg-NW scaffold. Nuclei are stained blue. Bar = 100 µm. c, Representative picture of Connexin 43 and sarcomeric actinin expression on days 3 and 8. 6 NATURE NANOTECHNOLOGY

7 SUPPLEMENTARY INFORMATION Supplementary Methods Conductivity and topography measurements by AFM. Topography and conductivity measurements were performed using an Asylum Research AFM (model MFP-3D). We used Pt/Ir-coated probes (Veeco Probes, Camarillo, CA, SPM-PIC, 0.2 N/m spring constant, 13 khz resonant frequency). For topography and current maps, the sample was rastered in contact mode with a 200 mv tip bias. Topography and current were recorded simultaneously. For current vs. voltage curves, the tip was positioned either over a NW or alginate region. A triangular wave potential bias (4 cycles, 0.5 Hz, 1V amplitude) was applied to the tip, and the current was simultaneously recorded. Impedance measurements on thin films. Thin films were prepared by sandwiching alginate film (with or without NWs) between two ITOcoated slides (Sigma, Ω/sq) Subsequent AFM measurements revealed that the resulting films were ca. 500 nm thick. For impedance measurements, we applied an AC potential bias between the ITO electrodes and swept the frequency between 10 6 Hz and 1 Hz (0.1V amplitude, decade/sec sweep rate). The real and imaginary components of the impedance at each frequency were recorded. Below 10 3 Hz, the pristine alginate film was not sufficiently conductive to yield reliable measurements (data not shown). Cardiac patch construction, cultivation and analysis. The cardiac patch was prepared as previously described 4. Briefly, cardiac cells were isolated from the left ventricles of SD neonatal (0-1 day old) rat hearts and seeded onto either Alg-NW or alginate scaffolds (5 x 2 mm, d x h, 0.7x10 8 cells/cm 3 ). The cell-seeded constructs were cultured for 3 days in normal conditions (humidified incubator 5% CO 2, 37 0 C, no electrical field). At that NATURE NANOTECHNOLOGY 7

8 point, constructs (both with NWs and pure alginate) were subjected to electrical stimulation (rectangular, 2 ms, 5 V/cm, 1 Hz) as previously described 6. Briefly, the constructs were cultivated in a glass chamber fitted with two 1/4-inch-diameter carbon rods (Ladd Research Industries, Burlington, VT) placed 1 cm apart and connected to a cardiac stimulator (S88 Grass dual output square pulse stimulator, Astro-med Inc. RI) with platinum wires (Ladd Research Industries). At the end of cultivation period, the patches were analyzed for viability using the XTT assay as described previously 2 (n>4 for each data point, collected from 2 separate experiments), or stained. Mechanical Testing The viscoelastic properties of the hydrogels were measured using an AR-2000 rheometer (TA Instruments, Inc., New Castle, DE). The scaffolds in the wet state were tested at a rate of 10% strain/min on an Instron 5542 mechanical tester (Instron, Norwood, MA). The compressive modulus (Ec) was determined as the slope of the linear region corresponding with 0-5% strain. Histology and immunofluorescence For histology, the cellular constructs were dehydrated in graduated alcohol steps (70 100%), paraffin-embedded, cut into 5-mm-thick sections, and mounted on slides. The sections were stained with hematoxylin and eosin (H&E). For immunofluorescence, the cellular constructs were fixed and permeabilized in cold methanol, blocked for 1 h at room temperature in Dulbecco s modified Eagle s medium (DMEM)-based buffer containing 5% FBS. After three buffer washes, the samples were incubated for 1 h with anti-troponin I (AbCam, Cambridge MA) or connexin 43 (Invitrogen, Carlsbad, CA) antibodies. After incubation, the samples were washed and incubated for 1 h with secondary antibodies. For nuclear detection, the cells were 8 NATURE NANOTECHNOLOGY

9 SUPPLEMENTARY INFORMATION incubated for 3 min with Hoechst and washed. Imaging was performed with a DeltaVision RT deconvolution microscope using the 20 or 40 objective (Applied Precision Inc. Northwest Issaquah, WA). Western blotting Total extracted protein (50 mg) was size-fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The membranes were incubated with antibodies against Cx-43 (Invitrogen, Carlsbad, CA), sarcomeric actinin (Sigma) and normalized against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). At least four cell constructs were tested with each antibody. Densitometric analysis was carried out using ImageJ analysis software (NIH). NATURE NANOTECHNOLOGY 9