Poly(amidoamine) dendrimer nanocarriers and their aerosol formulations for siRNA delivery to the lung epithelium

Abstract

Small interfering RNA (siRNA)-based therapies have great promise in the treatment of a number of prevalent pulmonary disorders including lung cancer, asthma and cystic fibrosis. However, progress in this area has been hindered due to the lack of carriers that can efficiently deliver siRNA to lung epithelial cells, and also due to challenges in developing oral inhalation (OI) formulations for the regional administration of siRNA and their carriers to the lungs. In this work we report the ability of generation four, amine-terminated poly(amidoamine) (PAMAM) dendrimer (G4NH2)-siRNA complexes (dendriplexes) to silence the enhanced green fluorescent protein (eGFP) gene on A549 lung alveolar epithelial cells stably expressing eGFP. We also report the formulation of the dendriplexes and their aerosol characteristics in propellant-based portable OI devices. The size and gene silencing ability of the dendriplexes was seen not to be a strong function of the N/P ratio. Silencing efficiencies of up to 40% are reported. Stable dispersions of the dendriplexes encapsulated in mannitol and also in a biodegradable and water-soluble co-oligomer were prepared in hydrofluoroalkane (HFA)-based pressurized metered-dose inhalers (pMDIs). Their aerosol characteristics were very favorable, and conducive to deep lung deposition, with respirable fractions of up to 77%. Importantly, siRNA formulated as dendriplexes in pMDIs was shown to keep its integrity after the particle preparation processes, and also after long-term exposures to HFA. The relevance of this study stems from the fact that this is the first work to report the formulation of inhalable siRNA with aerosol properties suitable to deep lung deposition using pMDIs devices that are the least expensive and most widely used portable inhalers. This study is relevant because, also for the first time, it shows that siRNA-G4NH2 dendriplexes can efficiently target lung alveolar epithelial A549 cells and silence genes even after siRNA has been exposed to the propellant environment.

Figure 1

Size and morphology of siRNA–G4NH2 dendriplexes at N/P 20 as determined by LS (main distribution in the center), SEM (upper left inset), and AFM (lower left inset). Histogram and Gaussian fit to the diameter distribution obtained from SEM images (>400 particles) of the dendriplexes is also shown (upper right inset).

Figure 2

siRNA complexation efficiency (indirect measurement) as a function of the N/P ratio, as quantified by PicoGreen Assay of residual free siRNA in the dispersion after preparation of the dendriplexes. Inset: Nondenaturing agarose gel electrophoresis of the corresponding dendriplexes: N/P 0.2 (lane 2), 0.5 (lane 3), 0.8 (lane 4), 1 (lane 5), 2 (lane 6), 3 (lane 7), 5 (lane 8), 10 (lane 9), 20 (lane 10), 30 (lane 11). Untreated siRNA control (300 ng) is shown in lane 1.

Figure 3

RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before (lane 1) and after (lane 2) incubation with RNase A.

Figure 4

RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7, 8–11, 12–15, 16–19, 20–23, respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1, after incubation with heparin (lane 2) and 0.35 μg RNase A per 1 μg siRNA (lane 3).

Figure 5

In vitro release of siRNA from dendriplexes in 0.1 M citrate/phosphate buffer at pH 5 and 7.4 (mimicking intracellular endosomes/lysosomes and cytosol, respectively), at 37 °C for dendriplexes at N/P ratio (a) 10; (b) 20; and (c) 30.

Figure 6

In vitro cytotoxicity of (a) PAMAM G4NH2 alone, and (b) siRNA–G4NH2 dendriplexes at N/P 30 in increasing concentration of G4NH2 (and thus siRNA – both concentrations shown). MTS assay on A549 lung alveolar cell line. * = statistically significantly different compared to untreated cells as control; and no statistical significant difference (n.s.d.) compared to untreated cells as control (n = 7, One-Way ANOVA followed by Tukey’s posthoc test, p value <0.05).

Figure 7

In vitro knockdown of eGFP expression in A549 cells stably expressing eGFP. siRNA–G4NH2 dendriplexes were prepared with siRNA as received from the supplier at N/P 5, 10, 20, and 30; with lyophilized siRNA stored in HFA-227 (HFA, at 25 °C and saturation pressure of the propellant) and freezer at −20 °C (FRE, at 253 K) for 2 months, at N/P 20. Specificity of the knockdown (siRNA(+) sequence, anti-eGFP) is maintained by comparison to effects with the siRNA(−) sequence (scramble). Lipofectamine 2000 (LF) and Transfast (TF) were the commercial transfection reagents used as positive controls, and free siRNA was the negative control. G4NH2 concentration at N/P 30 corresponds to 1.95 μM, and siRNA concentration in all systems was 80 nM. *,▼,⧫ = statistically significantly different compared to eGFP A549 cells treated with free siRNA(+) as received; and ∇ = no statistically significant difference (n.s.d.) among N/P 5, 10, 20, and 30 prepared with siRNA(+) as received (minimum n = 3, One-Way ANOVA followed by Tukey’s posthoc test, p value <0.05).

Figure 8

Size and morphology of (a) mannitol and (b) CSLA engineered microparticles loaded with siRNA–G4NH2 dendriplexes at N/P 10 as determined by LS (main distribution on right) and SEM (lower left inset). Particles were dispersed in HPFP (2 mg × mL^–1) to perform LS, and after that, the HPFP was evaporated, and 1 mL DI-water was added to dissolve the mannitol or CSLA shell, and LS was performed again, but at this time, the size of the dendriplexes released from the mannitol (or CSLA) was measured (upper left inset). Nondenaturing agarose gel electrophoresis (upper right inset) showing the integrity of siRNA after its release from mannitol (or CSLA) shell and upon incubation of the dendriplexes in aqueous heparin solution (455 U per 1 μg siRNA) for 30 min at 37 °C. Untreated siRNA (250 ng) as positive control in lane 1; mixture of G4NH2, mannitol (or CSLA) and heparin (but no siRNA) as negative control in lane 2; siRNA–G4NH2 dendriplexes at N/P 10 loaded into mannitol (or CSLA) microparticles after incubation with aqueous heparin in lane 3.

Figure 9

Aerosol properties of pMDI formulations prepared with siRNA–G4NH2 dendriplexes at N/P 10 loaded into (a) mannitol and (b) CSLA microparticles. pMDI formulations at 2 mg particles (mannitol or CSLA) per 1 mL in HFA-227 at 25 °C, and saturation pressure of the propellant. siRNA concentration of 290–550 ng × mL^–1 for pMDI formulations prepared with mannitol loaded with dendriplexes, and 420–505 ng × mL^–1 for those prepared CSLA loaded with dendriplexes. AC, IP, and F refer to actuator, induction port and filter, respectively. Insets: Physical stability of freshly prepared pMDI formulations.