Peptide-siRNA Supramolecular Particles for Neural Cell Transfection

Abstract

Small interfering ribonucleic acid (siRNA)-based gene knockdown is an effective tool for gene screening and therapeutics. However, the use of nonviral methods has remained an enormous challenge in neural cells. A strategy is reported to design artificial noncationic modular peptides with amplified affinity for siRNA via supramolecular assembly that shows efficient protein knockdown in neural cells. By solid phase synthesis, a sequence that binds specifically double-stranded ribonucleic acid (dsRNA) with a self-assembling peptide for particle formation is integrated. These supramolecular particles can be further functionalized with bioactive sequences without affecting their biophysical properties. The peptide carrier is found to silence efficiently up to 83% of protein expression in primary astroglial and neuronal cell cultures without cytotoxicity. In the case of neurons, a reduction in electrical activity is observed once the presynaptic protein synaptophysin is downregulated by the siRNA-peptide particles. The results demonstrate that the supramolecular particles offer an siRNA delivery platform for efficient nonviral gene screening and discovery of novel therapies for neural cells.

Keywords: glial cells; glial fibrillary acidic protein (GFAP); knockdown; neurons; protein engineering; supramolecular particles; synaptophysin; transfection.

Figure 1

Design of peptide with amplified siRNA binding via self‐assembly. a) Scheme showing crystal structure of a dsRNA‐binding domain from X. laevis (see ref. 15) (left). C‐terminus RNA binding motif P2 (in yellow) was extracted from the dsRBD and fused to a self‐assembly enhancer peptide P3 to obtain P4, a peptide with amplified siRNA binding (right). Black marks on the peptide schematics are dsRNA‐binding sites according to the crystal structure. b) Electrophoresis gels of peptides bound to siRNA. c) Plot of percentage of peptide‐bound siRNA derived from the electrophoresis gels. d) Plot of molar ellipticity (circular dichroism) of P4 and P2 peptides alone (dashed lines) and with siRNA (solid lines). e) Hydrodynamic size of P4 peptide alone (dashed line) and with siRNA (solid line). f) P4–siRNA particles in solution imaged by cryo‐TEM. g) Schematic representation of an unstructured P4 polypeptide that binds siRNA to assemble into particles. h) ζ‐potential of P4 peptide alone (empty bar) and with siRNA (solid bar). i) Electrophoresis gels showing the release and digestion of siRNA bound to peptide P4 when incubated in different media, DMEM (top), FBS 100% (middle), and DMEM + FBS 10% (bottom). j) Electrophoresis gels showing the release of siRNA from siRNA–P4 particles in aqueous solutions as a function of NaCl concentration. k) Electrophoresis gel showing release of siRNA from P4 particles when incubated with proteinase K. Molecular marker (first lane from left to right), naked siRNA (second lane), siRNA–P4 complexes (third lane), and siRNA–P4 complexes incubated with proteinase K for 24 h (fourth lane).

Figure 2

siRNA transfection and protein knockdown of neural cells. a,b) Percentage of transfected cells (blue graph) and amount of internalized fluorescently labeled siRNA–Alexa488 per cell (green graph) in a) astroglial and b) neuronal cell cultures. c,d) Percentage of cell survival in c) astroglial and d) neuronal cell cultures 24 h after transfection. e,h) Percentage of e) astroglial and h) neuronal cells transfected with P4 particles after their preincubation with inhibitors of three endocytic pathways: chlorpromazine (chlorprom or C), an inhibitor of clathrin‐mediated intracellular transport; filipin (F), an inhibitor of cholesterol‐dependent caveolar cell transport; and amiloride (A), an inhibitor of Na⁺/H⁺ exchange in ligand‐independent, nonselective transport by macropinocytosis. f,i) Confocal images of f) astroglial i) and neuronal cells stained with Lysotracker (red, endosomes), siRNA–Alexa488 (green, siRNA), and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue, nucleus) 24 h post‐transfection. g,j) Graph representing the Pearson's coefficient for siRNA/Lysotracker colocalization in g) astroglial and j) neuronal cell culture (−1 ≤ Rr ≤ 1, where 1 = maximum colocalization and −1 = maximal exclusion). k) Confocal images of astroglial cells stained with GFAP (green) and neuronal cells stained with synaptophysin (Syp, green), Tuj‐1 (red), and DAPI (blue) 24 h after transfection. l–o) Western blots and densitometry (intensity values normalized to actin) of l,m) GFAP and n,o) synaptophysin marker in neural cultures transfected with different P4 doses (doses 1, 2, 3 and 4 have concentrations of P4 supramolecular particles of 34 × 10⁻⁶, 52 × 10⁻⁶, 78 × 10⁻⁶, and 104 × 10⁻⁶ m, respectively, and of 65 × 10⁻⁹, 100 × 10⁻⁹, 150 × 10⁻⁹, and 200 × 10⁻⁹ m for siRNA, respectively) 24 h post transfection. *P < 0.05, ** P < 0.01, and **P < 0.001, least significant difference test (LSD) test (n = 7). All the experiments were performed using a dose 4 (P4: 104 × 10⁻⁶ m, siRNA: 200 × 10⁻⁹ m) except for western Blot analysis where four doses were used.

Figure 3

Functionalization of P4 supramolecular particles. a) Schematic representation of P4 peptide functionalized with bioactive peptides His6 (red) and RGD (blue). b) Plot of percentage of bound siRNA by P4, P4–RGD, and P4–His6 peptides calculated from a gel electrophoresis assay. c) Circular dichroism of P4, P4–His6, and P4–RGD peptides alone (dashed lines) and with siRNA (solid lines). d) Hydrodynamic size of P4, P4–His6, and P4–RGD peptides alone (dashed lines) and with siRNA (solid lines). e) ζ‐potential of peptides alone (empty bars) and with siRNA (solid bars) (results of P4 taken from Figure 1 are included for comparison purposes). f) Cryo‐TEM of peptide particles coassembled with siRNA. g,h) Percentage of positive transfected cells (blue graphs) and amount of internalized fluorescently labeled siRNA–Alexa488 per cell (green graph) with functionalized peptide P4 particles in g) astroglial cell and h) neuronal culture. i,j) Graph showing the Pearson's coefficient values for siRNA/Lysotracker colocalization in i) astroglial and j) neuronal cell culture (−1 ≤ Rr ≤ 1, where 1 = maximum colocalization and −1 = maximal exclusion). k–n) Western blots and densitometry (intensity values normalized to actin) of k,l) GFAP marker and m,n) synaptophysin (Syp) after 24 h of transfection in P4 functionalized with His6 (P4–His6) and RGD (P4–RGD) with a negative (−) or positive (+) siRNA. *P = 0.05, **P = 0.001, LSD test, n = 8 (GFAP) and n = 6 (synaptophysin). All the experiments were performed using a dose 4 (P4: 104 × 10⁻⁶ m, siRNA: 200 × 10⁻⁹ m).

Figure 4

Electrophysiological studies of functional gene knockdown of neuronal cell cultures by supramolecular particles. a) Structured illumination microscopy (SIM) images of cortical neuronal cells stained with Tuj‐1 (red) and synaptophysin (Syp, green) 24 h after transfection. b) Concept scheme of multielectrode array plates (MEA plates) for neural cell recording. c) Raster plots of a single MEA plate well of representative control and P4–His6‐treated cells before transfection (day 0) and 72 h post‐transfection (day 3). Each line represents the signals detected by a single electrode of the MEA array, during 100 s of recordings. d) Longitudinal progression of spike firing frequency and e) burst number. f) Schematic representation of cells transfected by P4 particles. Two‐way repeated measures one‐way analysis of variance (ANOVA) were performed for firing frequency and burst number. *P = 0.05, **P = 0.001, and ***P = 0.0001. All the experiments were performed using a dose 4 (P4: 104 × 10⁻⁶ m, siRNA: 200 × 10⁻⁹ m).