Mechanisms of nucleotide trafficking during siRNA delivery to endothelial cells using perfluorocarbon nanoemulsions

Abstract

RNA interference (RNAi) is a useful in vitro research tool, but its application as a safe and effective therapeutic agent may benefit from improved understanding of mechanisms of exogenous siRNA delivery, including cell trafficking and sorting patterns. We report the development of a transfection reagent for siRNA delivery which employs a distinctive non-digestive mode of particle-cell membrane interaction through the formation of a hemifusion complex resulting in lipid raft transport of cargo to the cytosol, bypassing the usual endosomal nanoparticle uptake pathway. We further demonstrate markedly enhanced efficacy over conventional transfection agents for suppressing endothelial cell expression of upregulated vascular adhesion molecules.

Figure 1

Cationic perfluorocarbon nanoparticles. Liquid perfluorocarbon based nanoparticles are formulated with the cationic lipid 1,2-Dioleoyl-3-Trimethylammonium-Proane (DOTAP) and the fluorescent lipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Carboxyfluorescein) within the surrounding phospholipid-surfactant monolayer (illustrated in the cartoon and TEM image). Electrostatic interactions between the charged lipids of the nanoparticles and the negatively charged phosphate backbone of siRNA lead to the formation of transfection complexes.

Figure 2

Delivery of fluorescently labeled transfection complexes to 2F-2B mouse endothelial cells. Mouse endothelial cells were treated with 12.5 nM DY-547 fluorescently labeled siRNA (red) and 25 pM cationic perfluorocarbon nanoparticles (green). Confocal micrographs illustrate the interaction of transfection complexes with cells directly after the removal of free transfection complexes (2h), and delivery of siRNA across the plasma membrane 4h after transfection. The white outline (4h) indicates the cell membrane. Scale bar, 10 μm.

Figure 3

Effective siRNA mediated VCAM-1 knockdown with perfluorocarbon nanoparticles. Mouse endothelial cells were treated with transfection complexes (10 pM cationic nanoparticles + 5 nM siRNA) for 2h under standard cell culture conditions. 2F-2B cells transfected with 10 pM perfluorocarbon nanoparticles + 5 nM anti-VCAM-1 siRNA show a decrease in VCAM-1 mRNA level (28.1 ± 6.7%) vs. both irrelevant siRNA (81.8 ± 7.3%) and no treatment controls (100%). VCAM-1 protein levels at 48h post incubation follow the same trend (VCAM-1: 14.8 ± 0.7 %, irrelevant siRNA: 87.7 ± 4.9% and no treatment control: 100%) (A). Short term membrane permeability (viability) was measured after treatment via flow cytometry with TOPRO3. Loading of perfluorocarbon nanoparticles with siRNA exerts a protective effect on cell viability versus cationic particles alone. * denotes p < 0.05 vs. no treatment control using a two tailed student's t-test. Data are presented as average ± standard error.

Figure 4

Perfluorocarbon nanoparticle-based siRNA transfection efficiency exceeds that of Lipofectamine2000. 2F-2B cells were transfected with anti-VCAM-1 siRNA under the following conditions: 10 pM perfluorocarbon nanoparticles + 5 nM siRNA (PFC-NP), Lipofectamine2000 + 5 nM siRNA, or Lipofectamine2000 + 100 nM siRNA. VCAM-1 mRNA levels (graphed as average + standard error) determined by real time PCR indicated that the decrease in VCAM-1 mRNA level 48h post incubation with PFC-NP is significantly lower than that under both Lipofectamine2000 conditions (3.3 fold decrease vs. Lipofectamine2000 + 5 nM siRNA and 2.5 fold decrease vs. Lipofectamine2000 + 100 nM siRNA). Transfection complex size for perfluorocarbon nanoparticles and complexes of nanoparticles and siRNA are significantly smaller than those for the Lipofectamine2000 conditions. * denotes p < 0.05 vs. perfluorocarbon nanoparticles + 5 nM siRNA using a two tailed students t-test. † denotes p< 0.05 vs. Lipofectamine2000 alone. Data are presented as average ± standard error.

Figure 5

Mechanism of perfluorocarbon nanoparticle mediated siRNA transfection. 2F-2B cells were incubated with transfection complexes containing 12.5 nM DY-547 fluorescently labeled siRNA(red) and 25 pM cationic nanoparticles(green) for 2h at either 37°C, 17°C, or 4°C, showing that intracellular siRNA delivery is an energy requiring transport process. For co-localization studies, 2F-2B cells were treated with 12.5 nM DY-547 labeled siRNA(red) and with markers for macropinosomes (70kDa dextran), clathrin pits (transferrin), lipid rafts (CT-B, post stained), and caveosomes(CT-B). siRNA appears to interact with lipid rafts on the cell membrane, and to a more limited extent with caveosomes within the cells, but not with clathrin pits or macropinosomes.