Molecular modeling and in vivo imaging can identify successful flexible triazine dendrimer-based siRNA delivery systems

Abstract

This study aimed to identify suitable siRNA delivery systems based on flexible generation 2-4 triazine dendrimers by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with thermodynamic parameters calculated using molecular modeling. The siRNA binding properties of the dendrimers and PEI 25 kDa were simulated, binding and stability were measured in SYBR Gold assays, and hydrodynamic diameters, zeta potentials, and cytotoxicity were quantified. These parameters were compared with cellular uptake of the complexes and their ability to mediate RNAi. Radiolabeled complexes were administered intravenously, and pharmacokinetic profiles and biodistribution of these polyplexes were assessed both invasively and non-invasively. All flexible triazine dendrimers formed thermodynamically more stable complexes than PEI. While PEI and the generation 4 dendrimer interacted more superficially with siRNA, generation 2 and 3 virtually coalesced with siRNA, forming a tightly intertwined structure. These dendriplexes were therefore more efficiently charge-neutralized than PEI complexes, reducing agglomeration. This behavior was confirmed by results of hydrodynamic diameters (72.0 nm-153.5 nm) and zeta potentials (4.9 mV-21.8 mV in 10 mM HEPES) of the dendriplexes in comparison to PEI complexes (312.8 nm-480.0 nm and 13.7 mV-17.4 mV in 10 mM HEPES). All dendrimers, even generation 3 and 4, were less toxic than PEI. All dendriplexes were efficiently endocytosed and showed significant and specific luciferase knockdown in HeLa/Luc cells. Scintillation counting confirmed that the generation 2 triazine complexes showed more than twofold prolonged circulation times as a result of their good thermodynamic stability. Conversely, generation 3 complexes dissociated in vivo, and generation 4 complexes were captured by the reticulo-endothelial system due to their increased surface charge. Molecular modeling proves very valuable for rationalizing experimental parameters based on the dendrimers' structural properties. Non-invasive molecular imaging predicted the in vivo fate of the complexes. Therefore, both techniques effectively promote the rapid development of safe and efficient siRNA formulations that are stable in vivo.

Figure 1

Structures of flexible core triazine dendrimers (F) of generation number 2, 3 and 4. The monochlorotriazine from which the peripheral group (2) for generation 2 and 4 carries one hydroxyl group and two amines.

Figure 2

Equilibrated configurations (A) F2-2, (B) F3, (C) F4-2, and (D) PEI interacting with DsiRNA. Nucleic acids are represented as black ribbons and surface amines that carry a +1 charge are represented as spheres. Water molecules and counter ions are omitted for clarity.

Figure 3

Figure 3A: Complexation behavior of dendrimers as measured by SYBR Gold intercalation of residual free siRNA at increasing N/P ratios. 3B: Release profiles of siRNA from polyelectrolyte complexes at N/P 5 as function of the concentration of heparin.

Figure 4

Hydrodynamic diameters and zeta potentials of dendrimer/siRNA complexes in comparison to PEI complexes as a function of solvent and N/P ratio.

Figure 5

Figure 5A. Confocal images showing the subcellular distribution of complexes made of Tye543-labeled siRNA (red) following cellular uptake in HeLa/Luc cells 4 hours after transfection. DAPI-stained nuclei are shown in blue. 5B-D. Knockdown of luciferase expression by dendrimer-siFLuc complexes in HeLa/Luc cells in comparison to dendriplexes with siNegCon (**p< 0.01, ***p< 0.001).

Figure 6

Three-dimensional biodistribution of A. F2-2-siRNA-dendriplexes, B. F3-siRNA-dendriplexes, and C. F4-2-siRNA-dendriplexes 2 hours after i.v. administration as registered by SPECT imaging.

Figure 7

Figure 7A. Biodistribution, 7B. Pharmacokinetics and AUC values in %ID*min/ml of siRNA-dendriplexes and polyplexes as measured by gamma scintillation counting of organ and blood samples.

Scheme 1

Hypothesized interaction of rigid polycations with siRNA leading to complexes of charged patches and flexible polycations yielding coalesced complexes of essentially neutralized charge.