Supporting Information

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1 Supporting Information Human Hepatocarcinoma Cell Targeting by Glypican-3 Ligand Peptide Functionalized Silica Nanoparticles: Implications for Ultrasound Molecular Imaging Marco Di Paola 1 *, Alessandra Quarta 2, Francesco Conversano 1, Enzo Antonio Sbenaglia 1, Simona Bettini 3, Ludovico Valli 4, Giuseppe Gigli 2 and Sergio Casciaro 1 1 Institute of Clinical Physiology, National Research Council, c/o Campus Ecotekne, via Monteroni, Lecce, Italy. 2 Institute of Nanotechnology, CNR-NANOTEC, c/o Campus Ecotekne, via Monteroni, Lecce, Italy. 3 Department of Engineering for Innovation, University of Salento, c/o Campus Ecotekne, via Monteroni, Lecce, Italy. 4 Department of Biological and Environmental Sciences and Technologies, University of Salento, c/o Campus Ecotekne, via Monteroni, Lecce, Italy. * m.dipaola@ifc.cnr.it; Tel: ; Fax: Contents Figure S1: HPLC and ESI-MS analysis of GPC-3 ligand peptide Figure S2: Experimental setup for ultrasound detection of SiNPs Figure S3: Size distribution and fluorescence spectra of naked and GPC-3 functionalized SiNPs Figure S4: Qualitative and quantitative estimation of peptide release Figure S5: Ultrasound imaging of SiNPs Figure S6: TEM analysis of HepG2 cells treated with SiNPs Figure S7: Confocal analysis of cell surface GPC-3 protein expression

2 Figure S1. HPLC (A) and ESI-MS (B) analysis of GPC-3 ligand peptide. The peptide was characterized by reversed-phase HPLC (Waters Alliance 2695 equipped with a diode array detector) coupled with an ESI-MS detector (Micromass ZQ, Waters, Milford Massachusetts, USA), using a Phenomenex Kinetex C 18 column (2.6 μm, mm) at 35 C. The solvent systems were the following: A (0.1% TFA in Milli-Q ultrapure H 2 O) and B (0.1% TFA in 84% CH 3 CN), with a flow of 0.6 ml/ min and a gradient of 20 70% B in 5 min. The peptide was detected as a single chromatographic peak at 4.01 min (Fig. S1A), and as [M+H] ion at m/z in the mass spectrum (Fig. S1B).

3 Figure S2. Scheme of the experimental setup for ultrasound detection of SiNPs. Agarose gel samples were imaged employing a digital echograph (Megas GPX, Esaote Spa, Florence, Italy), equipped with a linear probe (LA523, Esaote Spa) operating at 10 MHz, and linked via an optic fiber to a prototype platform for acquisition of unprocessed radiofrequency (RF) data (FEMMINA, ELEN Spa, Florence, Italy). The automatic scanning of the samples (scansion speed = 12.5 mm/min) was achieved by mounting the ultrasound probe on the motorized mechanism of an infusion pump (KDS 100, KD Scientific Inc., Holliston, MA). Each sample was fixed at the bottom of a water tank and the probe, also partially immersed in the water, was positioned perpendicular to the agarose surface at such a distance that the probe focus (set to 4 cm) was centered at the middle of the nanoparticle-containing gel layer. 100 frames of RF data were acquired in the central part of each scanned sample. The obtained RF frames were off-line processed through Fortezza software (Elen Spa, Florence, Italy) on a dedicated PC, in order to obtain time-dependent ultrasound backscatter amplitude of the acquired echographic images. For each acquired frame, a rectangular region of interest (ROI) composed of 25 ultrasound tracks with 250 points/track was set manually in the central zone of the echographic image, corresponding to the nanoparticle-containing gel, to avoid any boundary effects on the backscatter values. The ultrasound backscatter amplitude of each sample was the average of 100 values resulting from the corresponding frame sequence.

4 Figure S3. Size distribution (A) and fluorescence spectra (B) of SiNPs. In both panels, black lines and red lines correspond to naked and GPC-3 functionalized SiNPs, respectively. In (B) excitation wavelength was 494 nm.

5 Figure S4. Qualitative and quantitative estimation of peptide release. The upper panels refer to the UV absorption curves of naked (left upper panel) and functionalized (right upper panel) SiNPs, respectively. The samples were kept at 37 C for either 24, 48, 72 or 96 h, prior to be centrifuged, as described in the experimental section, and analyzed. In both panels, the dark red curve is related to nanoparticle samples treated with protease overnight at 37 C to induce full peptide release. The lower panel reports the quantitative analysis carried out on the collected filtrates by means of BCA assay. Data are the mean ± SD of two independent experiments, each in triplicate. Error bars represent SDs. Figure S5. Ultrasound imaging of SiNPs. Typical ultrasound images of phantoms containing: pure agarose (A), 0.25 mg/ml (B) and 0.5 mg/ml GPC-3 SiNPs (C). Red rectangles indicate the manually selected region of interest (ROI). D: ultrasound backscatter amplitude within the considered ROI for pure agarose (Ctrl) and the two indicated concentrations of SiNPs. Data are the mean ± SD of 100 analyzed frames. *: p 0.05 vs control.

6 Figure S6. TEM analysis of HepG2 cells treated with SiNPs. Binding and uptake of GPC-3 ligand peptide functionalized SiNPs after 1, 6, and 24 h incubation are shown in left panels. The correspondent images of naked SiNPs are shown in right panels. Typical images from two different experiments are reported. Scale bars = 500 nm.

7 Figure S7. Confocal analysis of cell surface GPC-3 protein expression. Dark (left panels) and correspondent bright field (right panels) confocal images of HepG2, MG-63, MDA and SKOV-3 cells treated with red-fluorescent, anti-gpc-3 monoclonal antibody. Scale bars = 75 μm. Typical images from two different experiments are reported.