Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

Hosam G Abdelhady,Yen-Ling Lin,Haiping Sun,Mohamed E H Elsayed,Imed Eddine Gallouzi

doi:10.1371/journal.pone.0061710

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.

Highlights

Preclinical investigations showed the potential of small interfering RNA molecules in selectively silencing the expression of the genes implicated in the development of cancer, cardiovascular, neurodegenerative, and infectious diseases indicating their therapeutic potential [1,2,3,4]. siRNA molecules bind to the RNAinduced silencing complex (RISC) revealing the antisense RNA strand that selectively binds to the complementary sequence in the targeted mRNA, which triggers mRNA cleavage by the endonuclease RNase H enzymes and suppression of the translation process [5,6,7]
One key advantage of atomic force microscopy (AFM) compared to other high resolution microscopy techniques is simple sample preparation that does not involve negative staining or shadow casting with a metal coating, which allows direct measurements that reflect the natural surface of the specimen [35,36,37]
We capitalized on established AFM protocols to image free siRNA molecules, G4 and G5 dendriplexes in liquid, and their susceptibility to enzymatic attack

Summary

Introduction

Preclinical investigations showed the potential of small interfering RNA (siRNA) molecules in selectively silencing the expression of the genes implicated in the development of cancer, cardiovascular, neurodegenerative, and infectious diseases indicating their therapeutic potential [1,2,3,4]. siRNA molecules bind to the RNAinduced silencing complex (RISC) revealing the antisense RNA strand that selectively binds to the complementary sequence in the targeted mRNA, which triggers mRNA cleavage by the endonuclease RNase H enzymes and suppression of the translation process [5,6,7]. Lipids, and polymers have been used to condense siRNA via electrostatic interaction forming ionic complexes with variable size and surface charge, which proved effective in delivering the RNA cargo into the cytoplasm of mammalian cells in vitro [8,9,10,11,12]. Successful in vivo delivery of siRNA required the use of excess cationic carrier to shield and protect the RNA cargo against nucleases leading to non-specific distribution of the formed complexes to the reticular endothelial system (liver, spleen, and bone marrow) [13] and induction of toxicity [14], which hampered the translation of these particles into the clinic. We are interested in formulation of compact dendriplexes that resist degradation by RNase enzymes without using excess PAMAM dendrimers to eliminate the associated toxicity

Methods

Results

Discussion

Conclusion