Supporting information Delivery of SiC-Based Nanoparticles into Live Cells Driven by Cell-Penetrating Peptides SAP and SAP-E

Preparation of the SiC-derived nanoparticles (SiC-NPs). SiC-based NPs were formed as describer earlier[S1] by means of electrochemical anodization of a low resistivity grade (<1 .cm) bulk 3C-SiC polycrystalline wafer. The etching process was performed for 3 hours at a current density of 25 mA/cm2 using a 1:1 HF (50%)/ethanol electrolyte. After the etching, a powder mixture composed mainly of carbon fluoroxide (CFO) NPs and 3C-SiC porous nanostructures was formed. The nano-powder was then naturally dried, removed from the SiC wafer, mechanically grinded and then dispersed in a Krebs buffer solution. The formed colloidal suspension was centrifuged at 10000 g for 5 minutes in order to collect only its top part containing very small (<10 nm) and homogeneously dispersed NPs which can be visualized by atomic force microscope (AFM) as shown in Figure S1-a. Size distribution of the obtained SiC-NPs was estimated from dynamic light scattering measurements (see Figure S1-b) and the average NPs size was found to be in the range of 4-6 nm. FTIR spectrum shown as right insert in Figure S1-b gives an idea about the chemical bonds (C-H, C=O and C-O) in the fabricated NPs. In addition, an elemental composition of the nanoparticles can be described as C100.0H104.1F19.5O51.0 brutto formula. The main surface chemical groups of the SiC-NPs are suggested to be carboxylic and ester groups. A UV-vis absorption spectrum of a colloidal suspension of the SiC-NPs in water is described in[S2]. Photoluminescence spectra of the SiC-NPs are found to be centered at 550 nm under excitation at 400 nm.


Supporting information
Delivery of SiC-Based Nanoparticles into Live Cells Driven by Cell-Penetrating Peptides SAP and SAP-E Tetiana Serdiuk, Iuliia Bakanovich, Vladimir Lysenko, Sergei A. Alekseev, Valery A. Skryshevsky, Sergii Afonin, E. Berger, Alain Géloën and Igor V. Komarov Preparation of the SiC-derived nanoparticles (SiC-NPs).SiC-based NPs were formed as describer earlier [S1] by means of electrochemical anodization of a low resistivity grade (<1  .cm) bulk 3C-SiC polycrystalline wafer.The etching process was performed for 3 hours at a current density of 25 mA/cm2 using a 1:1 HF (50%)/ethanol electrolyte.After the etching, a powder mixture composed mainly of carbon fluoroxide (CFO) NPs and 3C-SiC porous nanostructures was formed.The nano-powder was then naturally dried, removed from the SiC wafer, mechanically grinded and then dispersed in a Krebs buffer solution.The formed colloidal suspension was centrifuged at 10000 g for 5 minutes in order to collect only its top part containing very small (<10 nm) and homogeneously dispersed NPs which can be visualized by atomic force microscope (AFM) as shown in Figure S1-a.Size distribution of the obtained SiC-NPs was estimated from dynamic light scattering measurements (see Figure S1-b) and the average NPs size was found to be in the range of 4-6 nm.FTIR spectrum shown as right insert in Figure S1-b gives an idea about the chemical bonds (C-H, C=O and C-O) in the fabricated NPs.In addition, an elemental composition of the nanoparticles can be described as C 100.0 H 104.1 F 19.5 O 51.0 brutto formula.The main surface chemical groups of the SiC-NPs are suggested to be carboxylic and ester groups.A UV-vis absorption spectrum of a colloidal suspension of the SiC-NPs in water is described in [S2] .Photoluminescence spectra of the SiC-NPs are found to be centered at 550 nm under excitation at 400 nm.S3] Initial porous SiC-based nanopowder (10 mg) was refluxed for one hour in a mixture of ethylenediamine with anhydrous o-xylene (0.4 mL of En and 1.6 mL of o-xylene).Then the liquids were slowly distilled off (in the stream of N 2 , the bath temperature did not exceed 160 °C), afterwards Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2015 approx. 1 mL of o-xylene was added and distilled off to remove the residues of En and H 2 O from the reaction mixture.Brown waxy residue was re-dispersed in diluted water HCl solution under sonication and extracted several times by n-butanol to remove the excess of oxylene and other possible organic admixtures.Then the excess of Na 2 CO 3 solution was added to the remaining acidic solution, the SiC-NPs(+) were extracted in n-BuOH and large SiC crystallites were removed by centrifugation at 10 000 g for 5 minutes.Afterwards the SiC-NPs(+) were re-extracted into 0.01 M HCl, the obtained solution was diluted by Dulbecco's Modified Eagle's Medium (DMEM).According to zeta-potential measurements (pH 7), the as-prepared SiC-NPs(-) demonstrated sufficiently high negative zeta-potential close to -30 mV, which was supposedly caused by dissociation of the surface acidic -COOH and possibly Si-OH groups.Protonation of the grafted amino groups in aqueous solution resulted in the positive surface charges.The sample of aminated SiC-NPs(+) synthesized in large excess of En demonstrated high (+100 mV) zeta potential, because according to the FT-IR data, most of the acidic groups in this sample were consumed in the reaction with ethylenediamine, giving a surface covered mainly by NH 2 groups.

Treatment of the SiC-NPs with the cell-penetrating peptides SAP and SAP-E.
SiC-NPs(-) and SiC-NPs(+) (4 mg/mL in water) were mixed with SAP (4 mg/mL in water) and SAP-E (4 mg/mL in water) respectively, left standing for 3 hours before exposition to cell cultures.

Real-time cell analysis.
3T3-L1 cells were seeded at high density 20 000 cells/well for confluence study or at low density 2500 cells/well for proliferation assays in either E-plates 96.Cells were grown at 37°C in 5% CO 2 in DMEM 10% FCS with high glucose, and removed using trypsin 0.05% (PAA Laboratories, Les Mureaux, France).Cell proliferation and/or survival was monitored with the xCELLigence Real-time Cell Analyser (RTCA) System (ACEA Biosciences, Inc., San Diego, USA), which allows label-free monitoring changes of cell number, viability, morphology and quality of cell attachment by measurement of cell to electrode responses of cells seeded in E96-well plates manufactured with integrated microelectronic sensor arrays.RTCA system measures cell surface occupancy, i.e. cell index, taking into account cell number, cell size, and adhesion force.The results are represented as cell indexes (representative results are shown in Figure S2).The results showed that SAP, SAP-E and their complexes with the SiC-NPs in different combinations at the concentration 0.4 mg/mL have no significant effect on cell proliferation or on the cell death.Gene expression measurements.

Figure S1 :
Figure S1: a) AFM image of the used SiC-NPs; b) size distribution of the SiC-NPs, left insert shows colloidal aqueous solution with the dispersed SiC-NPs and right insert gives an idea on their dominating chemical bonds.

Figure S2 :
Figure S2: SAP or SAP-E were added to proliferating 3T3-L1 cells (arrow) (A).No significant effect of SAP or SAPE can be seen on cell index of the proliferating cells.3T3-L1 cells were seeded at high density to reach confluence (B).Addition of SAP or SAPE at 0.4 mg/mL is shown by the arrow.No significant effect of SAP or SAPE can be seen on the cell index.