Title

 

Authors

D. De Stefano¹, F. Ungaro², C. Giovino², A. Polimeno¹, F. Quaglia², R. Carnuccio^1
 
1Dept. of Experimental Pharmacology, University of Naples Federico II, Via D. Montesano, 49, 80131-Naples, Italy;
²Dept. of Pharmaceutical and Toxicological Chemistry, University of Naples Federico II, Via D. Montesano, 49, 80131-Naples, Italy;

 

Abstract

Cystic fibrosis (CF), an autosomal recessive genetic disorder, is caused by mutations within the cystic fibrosis transmembrane conductance regulator gene encoding for chloride channel protein, expressed on epithelial cells of various organs.¹ Morbidity and mortality depend on abnormal mucus secretion, persistent inflammatory response and chronic infections in the airways of CF patients.² P. aeruginosa, an opportunistic pathogen, chronically infects the lungand triggers a signalling pathway leading to NF-kB activation via Toll-like receptors.³NF-kB has been reported to regulate the expression of several genes encoding for pro-inflammatory mediators.⁴ These contribute to the overproduction of mucus/mucins, responsible for airway obstruction and mucociliary function failure in CFpatients.⁵ P. aeruginosaLPS induces the expression of MUC2 gene, whose promoter region contains consensus sequences to NF-kB.⁶ Thus, NF-kB may be a potential target for controlling lung mucus secretion and chronic inflammation in CFpatients. Oligonucleotides (ODN) as decoy cis-elements block the binding of NF-κB to promoter regions of its targeted genes, resulting in the inhibition of gene transactivation in vitro and in vivo and even in humans.^7-9 Biodegradable poly(DL-lactide-co-glycolide) (PLGA) microparticles allow an increase of ODN biological stability and prolonged delivery.⁸  We have recently demonstrated that NF-kB decoy ODN released from respirable large porous particles (LPP) of PLGA reduces IL-6 and IL-8 expression as well as NF-kB/DNA binding activity in LPS-stimulated CF human bronchial cellsat lower concentrations and for longer time-frames as compared to naked decODN.¹⁰The use of poly(ethyleneimine) (PEI) as adjuvant carrier of PLGA for ODN pulmonary delivery has been proposed since PEI is osmotic, bactericidal, and potentially mucolytic.¹¹ Here, we investigated the effects of NF-kB decoy ODN (decODN), entrapped into respirable PEI/PLGA LPP (PEI/LPP), on MUC2 expression as well as NF-kB/DNA binding activity in human lung NCI-H292 cells. The human lung NCI-H292 cells, over-expressing MUC2 gene, were stimulated with LPS (10 mg/ml) from P. aeruginosa for 24 and 72 h. Naked decODN, decODN-loaded PEI/LPP, scramble decODN-loaded PEI/LPP and PEI/LPP alone were added to the cells 10 min before LPS challenge at a final ODN concentration of 2.0 mM. Western blot, Polymerase Chain Reaction and Electrophoretic Mobility Shift Assay were performed as previously described.¹² Stimulation of NCI-H292 lung cells with LPS caused an increase of MUC2 mRNA and protein expression which was significantly inhibited by decODN-loaded PEI/LPP at 24 and 72 h, whereas naked decODN inhibited MUC2 mRNA and protein expression only at 24 h. Similar effects were elicited by decODN-loaded PEI/LPP and naked ODN on NF-kB/DNA binding activity in NCI-H292 lung cells stimulated with LPS at the same time points. In all of the experiments scramble decODN-loaded PEI/LPP as well as PEI/LPP alone had no effect. Our findings show that decODN-loaded PEI/LPP inhibit MUC2 expression as well as NF-kB/DNA binding activity in a more prolonged time as compared to naked ODN.Therefore, PEI/LPP could be an useful tool to improve pulmonary delivery of a decODN that, in turn, may represent a promising strategy to inhibit mucus production and chronic inflammation in CF patients.
 
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