Hybrid Lipid/Polymer Nanoparticles for Pulmonary Delivery of siRNA: Development and Fate Upon In Vitro Deposition on the Human Epithelial Airway Barrier

July 26, 2018

DOI: 10.1089/jamp.2017.1364

Ivana d’Angelo, PhD,1 Gabriella Costabile, PhD,2,3 Estelle Durantie, PhD,3 Paola Brocca, PhD,4 Valeria Rondelli, PhD,4 Annapina Russo, PhD,5 Giulia Russo, PhD,5 Agnese Miro, PharmD,2 Fabiana Quaglia, PhD,2 Alke Petri-Fink, PhD,3 Barbara Rothen-Rutishauser, PhD,3 and Francesca Ungaro, PhD2
1 Di.S.T.A.Bi.F., University of Campania ‘‘Luigi Vanvitelli,’’ Caserta, Italy.
2 Laboratory of Drug Delivery, Department of Pharmacy, University of Napoli Federico II, Napoli, Italy.
3 Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.
4 Applied Physics, Department of Medical Biotechnology and Translational Medicine, University of Milano, Milano, Italy.
5 Laboratory of Biochemistry, Department of Pharmacy, University of Napoli Federico II, Napoli, Italy.

The Vitrocell Cloud was used for the exposure to aerosolized nanoparticles on a in vitro three-dimensional cell cluture, mimicking the human epithelial airway barrier comprising human bronchial epithelial cells,human blood monocyte-derived macrophages and dendritic cells to show the optimized siRNA transport and delivery at lung.


Background: Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system.
Methods: A panel of DPPC/PLGA hNPs was prepared by emulsion/solvent diffusion and fully characterized. A combination of model siRNAs against the sodium transepithelial channel (ENaC) was entrapped in optimized hNPs comprising or not poly(ethylenimine) (PEI) as third component. siRNA-loaded hNPs were characterized for encapsulation efficiency, release kinetics, aerodynamic properties, and stability in artificial mucus (AM). The fate and cytotoxicity of hNPs upon aerosolization on a triple cell co-culture model (TCCC) mimicking human epithelial airway barrier were assessed. Finally, the effect of siRNA-loaded hNPs on ENaC protein expression at 72 hours was evaluated in A549 cells.
Results: Optimized muco-inert hNPs encapsulating model siRNA with high efficiency were produced. The developed hNPs displayed a hydrodynamic diameter of *150 nm, a low polydispersity index, a negative f potential close to -25 mV, and a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI. siRNA formulations showed optimal in vitro aerosol performance after delivery with a vibrating mesh nebulizer. Furthermore, small-angle X-ray scattering analyses highlighted an excellent stability upon incubation with AM, confirming the potential of hNPs for direct aerosolization on mucus-lined airways. Studies in TCCC confirmed that fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute proinflammatory effect. Finally, a prolonged inhibition of ENaC protein expression was observed in A549 cells upon treatment with siRNA-loaded hNPs. 
Conclusions: Results demonstrate the great potential of hNPs as carriers for pulmonary delivery of siRNA, prompting toward investigation of their therapeutic effectiveness in severe lung diseases.

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