Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.

The mucus obstructing the airways of Cystic Fibrosis (CF) patients is a yield stress fluid. Linear and non-linear rheological analyses of CF sputa can provide relevant biophysical markers, which could be used for the management of this disease. Sputa were collected from CF patients either without any induction or following an aerosol treatment with the recombinant human DNAse (rhDNAse, Pulmozyme^®). Several sample preparations were considered and multiple measurements were performed in order to assess both the repeatability and the robustness of the rheological measurements. The linear and non-linear rheological properties of all CF sputa were characterized. While no correlation between oscillatory shear linear viscoelastic properties and clinical data was observed, the steady shear flow data showed that the apparent yield stress of sputum from CF patients previously treated with rhDNAse was approximately one decade lower than that of non-treated CF patients. Similar results were obtained with sputa from non-induced CF patients subjected ex vivo to a Pulmozyme^® aerosol treatment. The results demonstrate that the apparent yield stress of patient sputa is a relevant predictive/prognostic biomarker in CF patients and could help in the development of new mucolytic agents.

The respiratory epithelium can be affected by many diseases that could be treated using aerosol gene therapy. Among these, cystic fibrosis (CF) is a lethal inherited disease characterized by airways complications, which determine the life expectancy and the effectiveness of aerosolized treatments. Beside evaluations performed under in vivo settings, cell culture models mimicking in vivo pathophysiological conditions can provide complementary insights into the potential of gene transfer strategies. Such models must consider multiple parameters, following the rationale that proper gene transfer evaluations depend on whether they are performed under experimental conditions close to pathophysiological settings. In addition, the mucus layer, which covers the epithelial cells, constitutes a physical barrier for gene delivery, especially in diseases such as CF. Artificial mucus models featuring physical and biological properties similar to CF mucus allow determining the ability of gene transfer systems to effectively reach the underlying epithelium. In this review, we describe mucus and cellular models relevant for CF aerosol gene therapy, with a particular emphasis on mucus rheology. We strongly believe that combining multiple pathophysiological features in single complex cell culture models could help bridge the gaps between in vitro and in vivo settings, as well as viral and non-viral gene delivery strategies.

New thioether-containing amphiphiles were designed, characterized and applied for gene delivery.

Aerosol lung gene therapy using non-viral delivery systems represents a credible therapeutic strategy for chronic respiratory diseases, such as cystic fibrosis (CF). Progress in CF clinical setting using the lipidic formulation GL67A has demonstrated the relevance of such a strategy while emphasizing the need for more potent gene transfer agents. In recent years, many novel non-viral gene delivery vehicles were proposed as potential alternatives to GL67 cationic lipid. However, they were usually evaluated using procedures difficult or even impossible to implement in clinical practice. In this study, a clinically-relevant administration protocol via aerosol in murine lungs was used to conduct a comparative study with GL67A. Diverse lipidic compounds were used to prepare a series of formulations inspired by the composition of GL67A. While some of these formulations were ineffective at transfecting murine lungs, others demonstrated modest-to-very-efficient activities and a series of structure-activity relationships were unveiled. Lipidic aminoglycoside derivative-based formulations were found to be at least as efficient as GL67A following aerosol delivery of a luciferase-encoding plasmid DNA. A single aerosol treatment with one such formulation was found to mediate long-term lung transgene expression, exceeding half the animal’s lifetime. This study clearly supports the potential of aminoglycoside-based cationic lipids as potent GL67-alternative scaffolds for further enhanced aerosol non-viral lung gene therapy for diseases such as CF.

A ramified lipid alcohol, 2-hexyldecanol, was used as a hydrophobic moiety to prepare cationic amphiphiles on a gram scale in 3 to 4 steps, featuring either a trimethylammonium 5, dimethylhydroxyethylammonium 6 or N-methylimidazolium 7 polar head group. Compression isotherms at the air–water interface reveal that all these cationic amphiphiles collapse at a relatively low pressure indicating a weak stabilization of the monolayer via hydrophobic interactions. Ellipsometry measurements point out the presence of a thin monolayer at low lateral pressure whereas thickening of the monolayer occurs at higher pressure with a high percentage of variation of the thickness, thus demonstrating an adaptability to the constraints. ³¹P NMR spectroscopy of the hydrated cationic amphiphiles clearly shows that these cationic amphiphiles self-assemble in water to form hexagonal phases, irrespective of the nature of their polar head group. Furthermore, a comparison of molecular structures suggests that compounds 5–7 self-organize into an inverted hexagonal phase (H_II). These cationic amphiphiles, alone or in the presence of DOPE, were evaluated for the transfection of three human-derived cell lines (i.e. A549, 16HBE and HeLa). The three compounds demonstrated high transfection efficacies in every cell line tested, 7/DOPE being the most efficient.