Visualizing the attack of RNase enzymes on dendriplexes and naked RNA using atomic force microscopy

Abstract

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/- ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/-) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.

Figure 1. Electrophoretic mobility of free siRNA, free G4 and G5 dendrimers, and the particles.

Image of the 1% w/v agarose gel stained with ethidium bromide showing the electrophoretic mobility of free siRNA, free G4 and G5 dendrimers, and the particles prepared by mixing G4 and G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at an N/P (+/−) ratio of 2/1 for 20 minutes or 24 hours.

Figure 2. AFM image of free siRNAs before and after attack by RNase enzyme.

(A) AFM image of free anti-GAPDH siRNA dissolved in 1 mM PBS containing 2 mM MgCl₂ after adding to the surface of freshly cleaved mica, which shows rod-, sphere-, and bead-like arrangements. (B) AFM image taken 1.5 minutes after adding RNase V1 enzyme, which shows rapid fragmentation of adsorbed siRNA molecules. (C) Time-lapse images showing a single siRNA molecule denoted by the white arrow (t = 0 min), the attack of RNase V1 enzyme on free siRNA molecule (t = 1.5 min), and complete siRNA degradation (t = 3 min). The scale bar in images A and B is 100 nm and the Z scale is 9 nm. The scale bar in image C is 35 nm and Z scale is 7 nm.

Figure 3. AFM image of G4 dendriplexes prepared for 20 minutes attacked by RNase enzyme.

(A) AFM image of hexagonal G4 dendriplexes prepared by mixing of G4 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 20 minutes at room temperature before loading onto the surface of freshly cleaved mica. AFM images of G4 dendriplexes after incubation with RNase V1 enzyme for 1–28 minutes (B–F) shows separation of the adsorbed dendriplexes and degradation of the complexed siRNA molecules (dark spots) that increased with the increase in incubation time. Two dendriplexes (defined wit dotted circles) remained intact throughout the incubation time with RNase V1 enzyme suggesting the formation of individual compact particles. Scale bar in these images is 200 nm and the Z scale is 15 nm.

Figure 4. AFM image of G4 dendriplexes prepared for 24 hours attacked by RNase enzyme.

Figure 5. AFM image of G5 dendriplexes prepared for 20 minutes attacked by RNase enzyme.

(A) AFM image of hexagonal G5 dendriplexes prepared by mixing of G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 20 minutes at room temperature before loading onto the surface of freshly cleaved mica. AFM images of G5 dendriplexes after incubation with RNase V1 enzyme for 1–60 minutes (B–F) shows separation of the adsorbed dendriplexes and degradation of the complexed siRNA molecules (dark spots) that increased with the increase in incubation time. Scale bar in these images is 200 nm and the Z scale is 17 nm.

Figure 6. AFM image of G5 dendriplexes prepared for 24 hours attacked by RNase enzyme.

(A) AFM image of G5 dendriplexes prepared by mixing of G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 24 hours at room temperature before loading onto the surface of freshly cleaved mica. G5 dendriplexes remain intact upon incubating with RNase V1 enzyme for 30 (B) and 60 minutes (C). Scale bar in these AFM images is 140 nm and the Z scale is 5 nm.