siRNA delivery to CNS cells using a membrane translocation peptide

Abstract

RNA interference (RNAi) is a sequence-specific gene silencing technique that has been applied to multiple pathological conditions. In this report, we describe the generation and in vitro characterization of an RNAi-based fluorescent probe for use as a therapeutic in the setting of ischemic stroke. Probe delivery to bEnd.3 brain endothelial cells and primary cortical neurons and astrocytes was promoted by incorporating small interfering RNA (siRNA) into complexes with fluorescently labeled myristoylated polyarginine peptides. The resulting probe was partially protected from serum nuclease degradation and was efficiently internalized by cells as confirmed by flow cytometry and confocal microscopy. In addition, application of the siRNA probe directed against c-Src, a protein implicated in stroke pathology, led to statistically significant reduction of endogenous c-src mRNA levels in all cell types tested. Results demonstrate the proof-of-principle that functionalized peptide--siRNA probes can be used as potential tools for dual imaging and therapeutic applications.

Figure 1

Gel mobility shift assay. MPAP-Cy5.5 was incubated with c-Src siRNA at several molar ratios to form MPAP-Src probes. At the 1:5 siRNA-to-MPAP ratio, most siRNA was incorporated into complexes.

Figure 2

Serum Stability. Stability of MPAP-Src (open squares) in serum relative to Dy547-labeled c-Src siRNA alone (closed squares) was investigated by incubating probes in 75% mouse serum for 0-8 hours. MPAP complexation to siRNA confers partial protection from serum nucleases, yielding a higher percentage of intact siRNA after incubation in serum than siRNA alone (mean±s.d.) relative to undigested controls (*).

Figure 3

Flow cytometry. Astrocytes, neurons and bEnd.3 cells were subjected to MPAP-Src or MPAP-ctrl incubation for 24 hours, followed by flow cytometry. All cell types were positive for the presence of both MPAP-Cy5.5 and siRNA-Dy547.

Figure 4

Confocal microscopy of astrocytes, neurons and bEnd.3 stained with GFAP (A), beta-III tubulin (B), and CD31 (C), respectively. Following incubation with MPAP-Src probe for 24 hours and cell-specific staining, cells were visualized by confocal microscopy. MPAP (D-F) and c-SRC siRNA (G-I) were taken up by cells following MPAP-Src incubation, whereas incubation with 100 pmoles of c-Src siRNA alone resulted in no significant siRNA uptake (M-O). Scale bar = 10 μm.

Figure 5

Cells were visualized using confocal microscopy following Lysosensor Green staining of unfixed astrocytes, bEnd.3 cells and neurons (D-F) to determine the mechanism of cellular uptake of MPAP-Src. MPAP (G-I) and c-Src siRNA (M-O) generally co-localize with endosomal vesicles. Scale bar = 10 μm.

Figure 6

Quantitative RT-PCR analysis of endogenous c-src mRNA expression levels (mean ±s.d., n = 7) in astrocytes, neurons and bEnd.3 cells treated with MPAP-ctrl (black bars) or MPAP-Src (gray bars) for 48 hours. After administration of MPAP-Src, there was a significant reduction in c-src levels in all cell types tested.

Figure 7

A. The percent survival (mean±s.d.) of cells treated with MPAP-Src or staurosporine (gray and white bars, respectively) relative to untreated controls (black bars) was obtained by MTT analysis. MPAP-Src treatment resulted in no significant increase in cytotoxicity in any of the cell types. B. Caspase-3 activity (mean±s.d.) in cells treated with MPAP-Src (gray bars) presented relative to activity in untreated cells (black bars). Though slight increases in caspase-3 signal were apparent, particularly in neurons, these were not statistically significant in any of the cells tested.