A retro-inverso cell-penetrating peptide for siRNA delivery

Background Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. Results Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. Conclusions This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins).Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down. Electronic supplementary material The online version of this article (doi:10.1186/s12951-017-0269-2) contains supplementary material, which is available to authorized users.

Fluorescence leakage assay was measured on a PTI spectrofluorometer at 25°C (Ex = 360 nm  3 nm; Em 530 nm  5 nm). In details, LUVs were diluted in 1 ml buffer (20 mM HEPES, 145 mM NaCl, pH 7.4) to a final concentration of 100 µM. To access the background fluorescence, the LUVs alone were measured S3 during 100 seconds. Thereafter, leakage was measured as an increase in fluorescence intensity upon addition of RICK or RICK-based nanoparticle (500 nM final RICK concentration) during the following next 900 seconds (15 min). Finally, 100% fluorescence was achieved by solubilizing the membranes with 0.1% (v/v) Triton X-100 resulting in the completely unquenched probe (at 1,000 seconds).

CPP adsorption at the air-water interface
"Monolayer techniques" are potent tools for studying the interfacial properties of membrane-active peptides in which the membrane hydrophobic-hydrophilic interface could be mimicked by an air-water interface or a monolayer of phospholipids as described (Konate et al. 2010;Deshayes et al. 2011). With this technique we carry out adsorption tests which consist in measuring surface pressure (Π) variations induced by increasing amounts of peptide at the air-water interface. Surface pressure was obtained at equilibrium, t = 30 minutes, after injection of aliquots of an aqueous solution of peptide into the aqueous sub phase (0.154 M NaCl solution), gently stirred with a magnetic stirrer. The maximal concentration for which no further variation of surface pressure is detected corresponds to the critical micellar concentration (CMC) indicative of the air-water interface affinity and amphipathic features of peptides.
Adsorptions at the air-water interface were performed with a homemade setup in which surface tension was measured with a Prolabo (France) tensiometer by using the platinum plate of the Wilhelmy method.

Tryptophan fluorescence spectrometry
Fluorescence experiments were performed on a PTI spectrofluorimeter at 25°C in 5% glucose. Intrinsic Tryptophan fluorescence of RICK at 5 µM was excited at 290 nm and emission spectrum was recorded between 320 and 390 nm, with a spectral band-pass of 2 and 6 nm for excitation and emission, respectively. LUVs of DOPC/SM/Chol (20:20:10, mol/mol/mol) were added to peptide solution at a final S4 lipid/peptide molar ratio of r = 80. All spectra are normalized to maximum of fluorescence of free peptides and plotted in relative fluorescence (%).

Atto633-labeling of the RICK peptide
For the Atto633 labeling, a RICK peptide bearing an N-terminal Cysteine-residue has been purchased from LifeTein. This peptide was reacted with a 2 molar excess of ATTO 633 maleimide (Atto Tec, Germany) during 5 h at room temperature. The Atto-peptide was then purified by RP-HPLC on a C18 column (Interchrom UP5 WOD/25 M Uptispere 300 5 ODB, 250 x 21.2 mm). The identity was assessed by Electrospray ionization mass spectra (Micromass Q-Tof, Waters).

Experiments on Giant Unilamellar Vesicles (GUVs)
GUVs were formed by using the hydration method (Weinberger et al, 2013). A 5% PolyVinylAlcohol, PVA (Sigma-Aldrich), solution is prepared (w/w) in ultra-pure water and homogeneously distributed over a coverslip (28 mm of diameter) which is left to dry for 30 min at 50°C. In the meantime, a lipids solution were prepared, composed in molar ratio of 1% No emission bleed through was observed between the different channels observed with these acquisition parameters. Emission photons were collected, through a 600 µm pinhole, on PhotoMultiplierTube (PMT) which allows an accurate photons counting. The obtained confocal images were projected and treated with the software ImageJ.

Confocal microscopy on U87 living cells:
The following acquisition parameters were chosen to record fluorescent NPs behavior inside living cells Size measurements using ImageJ indicated that big and small nanoparticles have a diameter of 120 ± 34 nm and 21 ± 6 nm, respectively. Scale bars correspond to 500 nm.

Mass spectra analyses
To confirm that the degradation products of CADY-K are due to the trypsin incubation, we collected the corresponding HPLC fractions for MS/MS analysis using a mass spectrometer (SYNAPT G2-S, Waters) equipped with an ESI source. Data acquisition was performed in positive ion mode in a mass range between 100 and 2000 Da. Interface parameters were set as follows: capillary voltage 3 kV and cone voltage 30 V. MS/MS spectra were recorded with cone voltage at 30 V and collision energy ramp between 15 eV and 60 eV was used. The data confirmed the expected digestion products of CADY-K.