Delivery of therapeutic AGT shRNA by PEG-Bu for hypertension therapy

Abstract

Gene silencing by RNA interference (RNAi) is a promising approach for gene therapy. However, up to today, it is still a major challenge to find safe and efficient non-viral vectors. Previously, we reported PEI-Bu, a small molecular weight PEI derivative, as an efficient non-viral carrier. However, like many PEI-based polymers, PEI-Bu was too toxic. In order to reduce cytotoxicity while maintain or even enhance transfecion efficiency, we modified PEI-Bu with poly(ethylene glycol) (PEG) to obtain PEG-Bu, and used it to delivery a theraputic short hairpin RNA (shRNA) targeting angiotensinogen (AGT) to normal rat liver cells (BRL-3A), which was a key target for the treatment of hypertension. The structure of PEG-Bu was confirmed by proton nuclear magnetic resonance ((1)H-NMR). Gel permeation chromatography (GPC) showed that the weight average molecular weight (Mw) of PEG-Bu was 5880 Da, with a polydispersity of 1.58. PEG-Bu could condense gene cargo into spherical and uniform nanoparticles with particle size (65-88 nm) and zeta potential (7.3-9.6 mV). Interestingly and importantly, PEG-Bu displayed lower cytotoxicity and enhanced tranfection efficiency than PEI-Bu after PEGylation in both normal cells BRL-3A and tumor cells HeLa. Moreover, PEG-Bu could efficiently delivery AGT shRNA to knockdown the AGT expression. To sum up, PEG-Bu would be a promising non-viral vector for delivering AGT shRNA to BRL-3A cells for hypertension therapy.

Figure 1. Reaction scheme of PEG-Bu.

Figure 2. Representative ¹H-NMR spectra of PEG-Bu.

Figure 3. Characterization of PEG-Bu/pDNA complexes.

A: Agarose gel electrophoresis of PEG-Bu/pDNA complexes at various w/w ratios. 1: Marker; 2: naked pDNA; 3–10: polymer/pDNA complexes at w/w ratios of 0.1,0.3,0.5,1,3,5,10,20. B: Particle size and zeta potential of PEG-Bu/pDNA complexes at various w/w ratios. C: Representative atomic force microscopic (AFM) image of PEG-Bu/pDNA complexes at a w/w ratio of 5.

Figure 4. Cytotoxicity of PEG-Bu.

Cytotoxicity of PEG-Bu at various concentrations and cytotoxicity of PEG-Bu/pDNA complexes at various w/w ratios in BRL-3A (A, B) and HeLa (C, D) cell lines. (n = 5, error bars represent standard deviation, *p<0.05, **p<0.01 vs PEI 25 kDa, ^Δp<0.05, ^ΔΔp<0.01 vs PEI-Bu).

Figure 5. Transfection efficiency of PEG-Bu.

Transfection efficiency of PEG-Bu/pGL3-Control complexes at various w/w ratios in serum–free media^, and transfection efficiency of PEG-Bu/pGL3-Control complexes (at optimal w/w) in serum-containing (10%) media in (A, C) BRL-3A, and (B, D) HeLa cell lines (n = 3, error bars represent standard deviation. As for A and B, *p<0.05, **p<0.01 vs PEI 25K, ^Δp<0.05, ^ΔΔp<0.01 vs PEI-Bu at optimal w/w 5).

Figure 6. Flow cytometry assay.

Transfection efficiency of PEG-Bu measured with flow cytometry in BRL-3A cells. A: fluorescence images observed with fluorescence microscopy. B: Representative histograms obtained from the flow cytometry, C: quantitative analysis of transfection efficiency of PEG-Bu as a percentage of GFP positive cells per total amount of BRL-3A cells in serum–free media. (n = 3, error bars represent standard deviation, **P<0.01 vs PEI 25K). Note: PEI 25K: PEI 25 kDa.

Figure 7. Gene silencing effect of PEG-Bu/shRNA (w/w 20) in BRL-3A cell lines.

A: Real-time PCR analysis of the effect of knockdown AGT expression. B: Western blot analysis of the effect of knockdown AGT expression. (**p<0.01 vs Blank control, ^ΔΔp<0.01 vs PEI 25 kDa/antisense shRNA).