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

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.

Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery

“…By taking into account the LNPs zeta potential which remained neutral for all LNP formulations in 5% lactose solution (m/V), LNPs are expected to pass the mucus layer without diffusion restrictions due to charge interactions. [ 60 ] As seen in Figure 7 , 65-90% of the freshly prepared LNPs penetrate the mucus layer, whereas only 20-40% of the spray dried LNP penetrate the mucus. This higher amount of penetration for freshly prepared LNPs can be explained by referring to the sizes of redispersed LNPs which lets us assume that the sugar matrix was not fully dissolved resulting in bigger nanoparticle sizes.…”

Non-Cellular Layers of the Respiratory Tract: Protection against Pathogens and Target for Drug Delivery

“…Bacteria were not present, which may explain why the advantage of PEGylation in cystic fibrosis mucus was not seen in another study by the same group. To deliver siRNA pool against the nuclear factor-κB (siNFκB), a non-PEGylated (DPPC) or PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG lipid shell was prepared [ 122 ]. The PEGylated particles did not better permeate cystic fibrosis sputum with polymicrobial colonization than the non-PEGylated carriers, whereas, on the other hand, the non-PEGylated constructs were ingested to a greater extent than the PEGylated carriers by Calu-3 and 16HBE14o- cells.…”

Can pulmonary RNA delivery improve our pandemic preparedness?

“…For our research, ALI cultures have become indispensable. While we can also measure mucus diffusion in simple cell-free setups using artificial or donor mucus in porous membrane chambers [ 69 ] or by fluorescence correlation spectroscopy [ 70 ], for example, only the actual cell culture experiment reflects the changes nanoformulations undergo in the presence of mucus, which may affect their cellular uptake. Even though pulmonary mucus does not contain serum, the formation of a protein corona in mucus has been discussed [ 71 ], and instabilities have been observed [ 72 ].…”

“…ALI models with different lung cancer and healthy lung epithelial as well as nasal epithelial cell lines have been established in my lab and routinely serve as models for assessing siRNA and drug delivery with nanoparticles through the mucus layer and into the epithelial cells and for the assessment of therapeutic gene silencing in ALI culture [ 69 , 71 ]. In this regard, my lab has mainly worked with monocultures, even if co-cultures with macrophages, dendritic cells or other cell types can of course add value in ALI models to better mimic the in vivo conditions [ 49 ].…”

“…While we mainly focused on anti-inflammatory siRNA delivery in the past and have recently reported on anti-NFkB siRNA delivery in a cystic fibrosis project in collaboration with Francesca Ungaro at the University of Napoli [ 69 ], we dove into new endeavors in the summer of 2019 and started collaborating with a young and upcoming virologist, Thomas Michler at TU Munich. At the time, he was interested in antiviral siRNA delivery to the lung to work on treatments of RSV.…”