Amino-acid functionalized porous silicon nanoparticles for the delivery of pDNA

Porous silicon nanoparticles as a novel platform in gene therapy, have shown to be an efficient vehicle for the delivery of nucleic acids in cells. For the first time, a family of porous silicon nanoparticles has been produced featuring an amino-acid functionalized cationic external surface aiming at pDNA complexation. The amino acid-based pDNA nanocarriers, displaying an average diameter of 295 nm, succeeded in transfection of HEK293 cells with an efficiency 300 times superior to “bare” porous silicon nanoparticles.


III. Characterizations of the functionalized nanoparticles
Particle sizes and size distributions were determined in ethanol by dynamic light scattering (DLS) using a Malvern Zetaziser particle sizer 380 (PSS) at a fixed angle of 90°. Infrared spectra were recorded on Nicolet IS5 spectrometer with the ATR ID5 module. UV-vis absorption measurements were performed using a lambda 35 Perkin Elmer spectrometer.
Transmission electron microscopy (TEM) images were obtained on JEOL 1200 EXII instrument.

III. 1. Zeta potential
Two drops of diluted functionalized porous silicon nanoparticles in absolute ethanol were added to 1 mL of absolute ethanol. The zeta measurements of the functionalized pSiNP were performed on Malvern Nanozetasizer.  Figure S2. Values recorded for zeta potential on the different nanostructures in ethanol. The negative charge value for pSiNP is due to the partial oxidation of the nanoparticles with the presence of silanol species at the surface of the particles. After the aminoacid grafting the charge of the systems switches to positive values whereas the pSiNP remains negative. This can be ascribed to the absence of amino-terminated groups beared by the lysine and histidine motifs.

III. 2. Dynamic light scattering
The DLS measurements of the materials were performed on a Malvern nanozetasizer in a diluted ethanol solution.  Figure S3. Size distribution by intensity of pSiNP and functionalized pSiNP.

III. 3. Infrared spectroscopy:
Infrared spectra were recorded on a Nicolet IS5 spectrometer with the ATR ID5 module. The narrow band centered at ≈800 cm -1 can be ascribed to the bending of NH/NH 2 1 , which is compatible with the presence of APTES (red) or aminoacid moieties (blue). On the other hand, at ≈2100 cm -1 is located the Si-H stretching band, which which is observed to decrease upon surface oxidation (vibronic Si-O-Si and Si-O (≈1150 cm -1 ) 2 and functionalization. Figure S4. DRIFTS spectra of the synthesized materials. pSiNp (brown), pSiNp@NH 2 (red) and both pSiNp@Lys and pSiNp@His (Blue).

Gel shift assay
Complexes formation between pSiNP and pDNA was evaluated by the gel-shift assay. Each pDNA/pSiNP dispersion with desired weight ratio was mixed with 6µl loading buffer bromophenol blue/ xylene cyanol) and then loaded on a 3% (W/V) agarose gel containing SYBR green dye. The amount of pDNA loaded into each well was 0.4 µg in a total volume of 10 µL. The electrophoresis was performed under 100V for 45 min. pDNA bands were visualized under UV irradiation at 304 nm (Gene Flash System transilluminator, Syngene mounted with Pulnix TM-300 camera).
The figure presents the combination of two pictures of two separate gels done at several nanoparticle concentrations explaining why backgroung appears different.

In vitro transfection experiments
Two days before transfection cells were seeded in 24 well culture plates density of 10 5 cells/cm². At the time of the transfection, the cells were 80 % confluent. Cells were then incubated for 4h at 37 °C with 0.5 mL (2.5 µg pCMV-luc) of the transfecting solution. Then, the medium was removed, replaced by fresh complete culture medium and the cells were cultured for 2 days before measurement of the luciferase activity.

Luciferase activity
Luciferase activity was measured using a lumat LB9507 luminometer (Berhold, Wildbach, Germany) according to Midoux and Mosigny 4 . The amount of proteins was determined by a modified bicinchoninic assay and the luciferase activity (relative light units, RLU) was normalized to RLU per miligram of extracted proteins.