Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles

Abstract

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.

Figure 1. Electrostatic interaction between CADY and HSPGs is not required for efficient transfection.

(A) HeLa cells were pre-treated for 30 min with free medium or medium containing 5 µg/ml heparin prior to addition of 80 nM siRNA complexed to CADY at a 1∶40 molar ratio. Cells were incubated in the presence of heparin for 1 hr, then extensively washed in PBS and replaced in DMEN containing 10% FCS. Cells were harvested after 48 hours and protein levels were analysed by Western blotting. CHO wild type (WT) (B), glycosaminoglycan deficient (gl^−/−) (C) and heparan sulfate deficient (HS^−/−) (D) cells were transfected with 80 nM FITC-labeled siRNA complexed to CADY at a 1∶20 molar ratio. After 1.5 hrs, cells were extensively washed, trypsinized and analyzed by FACS.

Figure 2. Cellular uptake of CADY:siRNA-FITC complexes in the presence of endosomal and lysosomal markers.

HeLa cells grown on glass coverslips were transfected with 80 nM FITC-labeled siRNA complexed to CADY at a 1∶4 ratio together with either choleratoxin subunit B (CtB) (A–C), transferrin (D–F) or lysotracker (G–L). Cells were extensively washed and fixed in PFA at indicated time points. (M–O) Non-transfected HeLa cells treated with CtB, Transferrin and Lysotracker.

Figure 3. Cellular uptake of CADY:siRNA-Cy3 complexes in the presence of endosomal markers.

HeLa cells grown on glass coverslips were transfected with 80 nM Cy3-labelled siRNA complexed to CADY at a 1∶4 molar ratio. After 1 hr, cells were fixed in PFA and stained with anti-caveolin (A–C) or anti-Rab5 (D–F).

Figure 4. CADY transfection in energy depleted cells.

HeLa cells were pre-incubated for 30 min at 37°C (A) or 4°C (B) prior to addition of 80 nM FITC-labeled siRNA complexed to CADY at a 1∶20 molar ratio. After 1.5 hr incubation at indicated temperatures, cells were washed, trypsinized and analysed by FACS. (C) HeLa cells were pre-treated with medium containing variable concentrations of sodium azide (NaN₃) for 30 mins prior to addition of 80 nM siRNA complexed to CADY at a 1∶40 molar ratio. Cells were incubated for 1 hr in the presence of NaN₃, then extensively washed, trypsinized and resuspended in medium containing 10% FCS. Cells were harvested after 48 hours and protein levels were analyzed by Western blotting.

Figure 5. CADY:siRNA complexes induce cellular influx of calcium.

HeLa cells were incubated with 5 µM FURA-2AM followed by washes in HEPES-Krebs-Ringer (HKR) buffer (A–C,E) or HKR buffer without calcium (D,E). The ratio of 340_{(Ca-bound FURA-2AM)}/380_(FURA-2AM) nm excitatory wavelengths was recorded upon addition of 20 nM siRNA complexed to CADY at a 1∶20 molar ratio. HeLa cells were transfected with 80 nM FITC labeled siRNA complexed to CADY at a 1∶20 molar ratio in the presence (F) or absence (G) of calcium. After 90 mins, cells were washed, trypsinized and analysed by FACS.

Figure 6. Electron micrographs of membrane interaction and internalization of CADY:siRNA nanoparticles.

HeLa cells were incubated with complexes of nanogold-labeled siRNA (80 nM) complexed to CADY at a 1∶20 molar ratio for 2 h. Interaction of CADY:siRNA-nanogold complexes as dense particles with plasma membrane (A, arrowheads in B), translocation into cells (arrows in B) and localization in endosomal vesicle (asterisk in C) or free in cytosol (arrows in C).