PAMAM Dendrimers for siRNA Delivery: Computational and Experimental Insights

Abstract

Short double-stranded RNAs, which are known as short interfering RNA (siRNA), can be used to specifically down-regulate the expression of the targeted gene in a process known as RNA interference (RNAi). However, the success of gene silencing applications based on the use of synthetic siRNA critically depends on efficient intracellular delivery. Polycationic branched macromolecules such as poly(amidoamine) (PAMAM) dendrimers show a strong binding affinity for RNA molecules and, hence, can provide an effective, reproducible, and relatively nontoxic method for transferring siRNAs into animal cells. Notwithstanding these perspectives, relatively few attempts have been made so far along these lines to study in detail the molecular mechanisms underlying the complexation process between PAMAMs and siRNAs. In this work we combine molecular simulation and experimental approaches to study the molecular requirements of the interaction of RNA-based therapeutics and PAMAM dendrimers of different generations. The dendrimers and their siRNA complexes were structurally characterized, and the free energy of binding between each dendrimer and a model siRNA was quantified by using the well-known MM/PBSA approach. DOSY NMR experiments confirmed the structural in silico prediction and yielded further information on both the complex structure and stoichiometry at low N/P ratio values. siRNA/PAMAM complex formation was monitored at different N/P ratios using gel retardation assays, and a simple model was proposed, which related the amount of siRNA complexed to the entropy variation upon complex formation obtained from the computer simulations.

Scaling trend in diffusion coefficients of low generation G0–G3 PAMAM dendrimers in aqueous solution at high and neutral pH

“…Recently, DOSY experiments were employed to determine the effect of palladium nanoparticles in the structure and diffusion properties of G4 and G6 PAMAM dendrimers, showing that these dendrimers do not form dendrimer metallic nanoparticles aggregates in solution [22,23]. In 2010, Pavan et al reported the diffusion coefficients of G4-G6 PAMAM dendrimers and their supramolecular complexes with siRNA, using a combined strategy of computational methods and DOSY experiments, showing that calculated structural parameters for PAMAM dendrimers at different pH were in excellent agreement with the actual NMR experiments [24]. Self-diffusion of lower generation G0-G3 PAMAM dendrimers has not been studied by experimental methods in aqueous solution.…”

Determination of spectral markers of cytotoxicity and genotoxicity using in vitro Raman microspectroscopy: cellular responses to polyamidoamine dendrimer exposure

“…Atomic simulations of dendrimer-nucleic acid interactions have been conducted by Nandy et al and the study showed the formation of stable DNA and siRNA complexes with PAMAM dendrimers at a physiological pH based on electrostatic interaction. 40 The strong binding affinity of PAMAM dendrimers to the RNA has also been shown in computational and experimental designs by 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pavan et al 41 The dose and time dependent changes on the nucleolus of the A549 cells upon PAMAM-G5 exposure are also evaluated in order to clarify mechanism of action of PAMAM-G5 dendrimers with RNA. The scatter plots of PCA of nucleolus of PAMAM-G5 exposed A549 cells and corresponding controls are provided in Supplementary Figure S9 and S10 for dose dependent and time dependent responses, respectively.…”

PAMAM dendrimer - cell membrane interactions

“…Modifications to the dendrimer surface, such as attaching sugar molecules (cyclodextrins) [39] and hydrophobic dye molecules (Oregon green 488) [40], have been found to improve transfection efficiency; however, the underlying transfection mechanisms are still unclear. However, the molecular mechanisms underlying complexation between short doubled stranded RNA (siRNA) and PAMAM dendrimers have been described by Pavan et al [41] using molecular simulation, NMR and electrophoresis assays. Electrostatic complexation of anionic DNA with cationic dendrimers has also been shown to result in the formation of DNA-linked PAMAM nanoclusters as described by MD simulations by Madal et al [42].…”