Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity

Abstract

A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.

Figure 1

Structures of triazine dendrimer cores and peripheral groups. The cores are labeled based on their degree of flexibility (rigid = G, bow-tie = B, and flexible = F) and generation number (2 or 3). The monochlorotriazines from which the peripheral groups were obtained are labeled based on the substitutions (hydroxyl groups and amines = 1 or 3, hydroxyl groups and guanidinylated amines = 1g or 3g, and alkyl chain and amines = 5).

Figure 2

Complexation behavior of dendrimers as measured by SYBR Gold intercalation of residual free siRNA at increasing N/P ratios.

Figure 3

In vitro stability of dendriplexes as measured by SYBR Gold intercalation of displaced siRNA at increasing concentrations of heparin.

Figure 4

Hydrodynamic diameters of dendrimer/siRNA complexes as determined by dynamic light scattering. The dendriplexes were formed at N/P ratio 5 at a concentration of 50 pmol siRNA in 50 μl of 5 % glucose.

Figure 5

Cellular uptake of dendriplexes made of Alexa488-labeled siRNA into HeLa/Luc cells after four hours of incubation as measured by flow cytometry.

Figure 6

Confocal images showing the subcellular distribution of dendriplexes made of Tye543-labeled siRNA (red) following cellular uptake in HeLa/Luc cells 4 hours after transfection. DAPI-stained nuclei are shown in blue.

Figure 7

Knockdown of luciferase expression by dendrimer-siRNA complexes in HeLa/Luc cells. Specificity of knockdown is maintained by comparison to effects of dendriplexes with the negative control sequence siNegCon. *p< 0.05, **p< 0.01, ***p< 0.001.

Figure 8A

Pharmacokinetics of vector and payload of siRNA-dendriplexes and polyplexes as measured by gamma scintillation counting of blood samples. 8B. Statistical analysis of the bioavailability of vector and load as measured by the area under the curve (AUC). ***p< 0.001

Figure 9

Biodistribution of vector and payload of siRNA-dendriplexes and polyplexes 2 hours after i.v. administration as measured by gamma scintillation counting of dissected organs.

Figure 10

Subcellular distribution of dendriplexes made of Tye543-labeled siRNA (red) in the lung and liver following i.v. administration. DAPI-stained nuclei are shown in blue.