Hybrid Lipid/Polymer Nanoparticles to Tackle the Cystic Fibrosis Mucus Barrier in siRNA Delivery to the Lungs: Does PEGylation Make the Difference?

Gemma Conte,Giulia Linardos,Raffaella Sorrentino,Paola Brocca,Rosaria Bassi,Francesca Ungaro,Gabriella Costabile,Olivia M Merkel,Valeria Rondelli,Ersilia V Fiscarelli,Domizia Baldassi,Diego Colombo,Agnese Miro,Fabiana Quaglia,Ivana D’Angelo

doi:10.1021/acsami.1c14975

Abstract

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air–liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o- cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

Highlights

ACS Applied Materials & Interfaces www.acsami.org that is based on core−shell nanoparticles comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell, exhibiting complementary characteristics of both polymeric nanoparticles and liposomes.[6−10] Of note, we recently demonstrated that the lipid layer surrounding the PLGA core likely confers muco-inertia to hybrid nanoparticles (hNPs), assisting the transport of the nucleic acid cargo through the mucus-covered human airway epithelial barrier.[11,12]
Since PEGylated nanoparticles are at the cutting edge to overcome the mucus barrier,[23] we examined the potential of PEGylated hNPs, which have been investigated so far for reasons other than small interfering RNA (siRNA) lung delivery.[24−27] siRNA loaded hNPs were prepared with a PLGA core and coated by either non-PEGylated or PEGylated lipid shells, employing dipalmitoyl phosphatidylcholine (DPPC) or N-(carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG)
■ RESULTS AND DISCUSSION Overall Properties of siRNA-Loaded hNPs. hNPs containing an siRNA pool against NFkB were successfully produced by an emulsion/solvent diffusion technique employing two different lipids as surface modifiers, that is DPPC and DSPE-PEG, in the presence

Summary

■ INTRODUCTION

Lung delivery of small interfering RNA (siRNA) holds great promise for the treatment of severe lung diseases, such as cystic fibrosis (CF).[1,2] As a matter of fact, synthetic therapeutic siRNA can be designed to virtually target any gene of interest with high selectivity, including those targets considered “undruggable”.3 the pulmonary route of administration offers the unprecedented opportunity to directly deliver siRNA to the diseased lung tissue in a loco-regional and minimally invasive manner.[1,4] despite the huge therapeutic potential, only two clinical trials providing for aerosolized siRNA have been undertaken till To understand the detailed role of AM components on the behavior of hNPs in mucus, the transport of fluorescent siRNA-loaded hNPs through AM was assessed by a Transwell multi-plate assay as previously reported (Figure 3A).[29] In line with previous literature data,[23] our results suggest that PEGylated hNPs allow for faster transport through AM as compared to non-PEGylated ones, with about 80% of the total amount of PEI/siNFkB_DSPE-PEGRhod hNPs permeated after 6 h versus 60% of PEI/siNFkB_DPPCRhod found in SILF at the same time intervals. Results confirm the ability of the developed hNPs to release siNFkB intracellularly and to induce in vitro inhibition of NFκB gene overexpression, likely preventing progressive and fatal degradation inside intracellular organelles (i.e., endosomes)

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES