Mucoactive drugs and multiple applications in pulmonary disease therapy.

Mucus is a complex polymeric hydrogel that serves as a critical defense in several organs. In the lungs, it provides a formidable barrier against inhaled particles such as microorganisms. In addition, mucus is essential for normal lung physiology, as it promotes immune tolerance and facilitates a normal commensal pulmonary microbiome. Hypersecretion of airway mucus is a characteristic of numerous respiratory diseases, such as Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF), and creates pulmonary obstruction, limiting the effectiveness of inhaled therapies. Due to those alterations, therapeutic strategies must be optimal to limit airway obstruction and restore pulmonary function. Mucoactive drugs are common therapeutic options and are classified into different groups depending on their modes of action, i.e., expectorants, mucokinetics, mucoregulators and mucolytics. This review focuses on mucoactive drugs and their modes of action. A special focus will be made on two challenging pulmonary pathologies: COPD and CF, and on their clinical studies conducted with mucoactive drugs.

BSV163/DOPE-mediated TRAIL gene transfection acts synergistically with chemotherapy against cisplatin-resistant ovarian cancer.

Ovarian cancer is the seventh most frequently diagnosed cancer among women worldwide. Most patients experience recurrence and succumb eventually to resistant disease, underscoring the need for an alternative treatment option. In the presented manuscript, we investigated the effect of the TRAIL-gene, transfected by an innovative bioinspired lipid vector BSV163/DOPE in the presence or absence of cisplatin, to fight against sensitive and resistant ovarian cancer. We showed that BSV163/DOPE can transfect ovarian cancer cell lines (Caov3, OVCAR3, and our new cisplatin-resistant, CR-Caov3) safely and efficiently. In addition, TRAIL-gene transfection in association with cisplatin inhibited cellular growth more efficiently (nearly 50% in Caov3 cells after the combined treatment, and 15% or 25% by each treatment alone, respectively) owing to an increase in apoptosis rate, caspases activity and TRAIL's death receptors expression. Most importantly, such synergistic effect was also observed in CR-Caov3 cells demonstrated by an apoptosis rate of 35% following the combined treatment in comparison with 17% after TRAIL-gene transfection or 6% after cisplatin exposition. These results suggest this combination may have potential application for sensitive as well as refractory ovarian cancer patients.

Impact of mucus and biofilm on antimicrobial photodynamic therapy: Evaluation using Ruthenium(II) complexes.

The biofilm lifestyle of bacterial pathogens is a hallmark of chronic lung infections such as in cystic fibrosis (CF) patients. Bacterial adaptation to the complex conditions in CF-affected lungs and repeated antibiotherapies lead to increasingly tolerant and hard-to-treat biofilms. In the context of growing antimicrobial resistance and restricted therapeutic options, antimicrobial photodynamic therapy (aPDT) shows great promise as an alternative to conventional antimicrobial modalities. Typically, aPDT consists in irradiating a non-toxic photosensitizer (PS) to generate reactive oxygen species (ROS), which kill pathogens in the surrounding environment. In a previous study, we reported that some ruthenium (II) complexes ([Ru(II)]) can mediate potent photodynamic inactivation (PDI) against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates. In the present work, [Ru(II)] were further assayed to evaluate their ability to photo-inactivate such bacteria under more complex experimental conditions better recapitulating the microenvironment in lung infected airways. Bacterial PDI was tentatively correlated with the properties of [Ru(II)] in biofilms, in mucus, and following diffusion across the latter. Altogether, the results obtained demonstrate the negative impacting role of mucus and biofilm components on [Ru(II)]-mediated PDT, following different possible mechanisms of action. Technical limitations were also identified that may be overcome, making this report a pilot for other similar studies. In conclusion, [Ru(II)] may be subjected to specific chemical engineering and/or drug formulation to adapt their properties to the harsh micro-environmental conditions of the infected respiratory tract.

Gene transfection using branched cationic amphiphilic compounds for an aerosol administration in cystic fibrosis context.

For cystic fibrosis gene therapy, the aerosolization of genetic materials is the most relevant delivery strategy to reach the airway epithelium. However, aerosolized formulations have to resist shear forces while maintaining the integrity of plasmid DNA (pDNA) during its journey from the nebulization to the epithelial cells. Herein, we compared the efficiency of gene delivery by aerosolization of two types of formulations: (i) BSV163, a branched cationic amphiphilic compound, co-formulated with different DOPE ratios (mol/mol) and DMPE-PEG5000 and (ii) 25 KDa branched polyethylenimine (b-PEI)-based formulation used as control. This study also aims to determine whether BSV163-based formulations possess the ability to resist the nebulization mechanisms and protect the nucleic acids (pDNA) cargo. Therefore, two CpG free plasmids (pGM144 or pGM169) encoding either the luciferase reporter gene or hCFTR respectively were used. Air-Liquid Interface (ALI) cell-culture was selected as an in-vitro model for aerosol experiments due to its closer analogy with in vivo morphology. Results highlighted that DOPE ratio influences the capacity of the BSV163 based-formulations to mediate high transfection efficacies. Furthermore, we proved that addition of DMPE-PEG5000 upon the formation of the BSV163/DOPE (1/1) lipid film instead of post-insertion led to a higher transgene expression. The aerosolization of this formulation on ALI cell-culture was more efficient than the use of b-PEI-based formulation.

Ruthenium(II) Polypyridyl Complexes for Antimicrobial Photodynamic Therapy: Prospects for Application in Cystic Fibrosis Lung Airways.

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.

Apparent Yield Stress of Sputum as a Relevant Biomarker in Cystic Fibrosis.

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.

Aerosol-Mediated Non-Viral Lung Gene Therapy: The Potential of Aminoglycoside-Based Cationic Liposomes.

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.

Optimizations of In Vitro Mucus and Cell Culture Models to Better Predict In Vivo Gene Transfer in Pathological Lung Respiratory Airways: Cystic Fibrosis as an Example.

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.

Branched lipid chains to prepare cationic amphiphiles producing hexagonal aggregates: supramolecular behavior and application to gene delivery.

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 (HII). 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.

Bis-Thioether-Containing Lipid Chains in Cationic Amphiphiles: Physicochemical Properties and Applications in Gene Delivery.

Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.

Substitution of unsaturated lipid chains by thioether-containing lipid chains in cationic amphiphiles: physicochemical consequences and application for gene delivery.

The hydrophobic moiety of cationic amphiphiles plays an important role in the transfection process because its structure has an impact on both the type of the supramolecular assembly and the dynamic properties of these assemblies. The latter have to exhibit a compromise between stability and instability to efficiently compact then deliver DNA into target cells. In the present work, we report the synthesis of new cationic amphiphiles featuring a thioether function at different positions of two 18-atom length lipid chains and we study their physicochemical properties (anisotropy of fluorescence and compression isotherms) with analogues possessing either oleyl (C18:1) or stearyl (C18:0) chains. We show that the fluidity of cationic lipids featuring a thioether function located close to the middle of each lipid chain is intermediate between that of oleyl- and stearyl-containing analogues. These properties are also supported by the compression isotherm assays. When used as carriers to deliver a plasmid DNA, thioether-containing cationic amphiphiles demonstrate a good ability to transfect human-derived cell lines, with those incorporating such a moiety in the middle of the chain being the most efficient. This work supports the use of a thioether function as a possible alternative to unsaturation in aliphatic lipid chains of cationic amphiphiles to modulate physicochemical behaviours and in turn biological activities such as gene delivery ability.