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.

Effects of the Sex Steroid Hormone Estradiol on Biofilm Growth of Cystic Fibrosis Pseudomonas aeruginosa Isolates.

Women with cystic fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization with biofilm formed by Pseudomonas aeruginosa. There is growing evidence that blood serum concentrations of the steroid sex hormone estradiol (E2) correlate with the occurrence of pulmonary exacerbations in CF but also play a role in the mucoid switch of P. aeruginosa. This study aims to shed light on possible microbiological reasons for sexual dimorphism in CF by investigating the influence of E2 on biofilm formation of P. aeruginosa CF isolates. For this purpose, 10 CF isolates of the respiratory tract derived from different CF patients have been treated with E2 in a microtiter plate biofilm model. Biofilms have been examined by crystal violet assays, field emission scanning electron microscopy (FE-SEM), 3D laser scanning microscopy (LSM), and quorum sensing (QS) reporter assays of the supernatants taken from biofilms. This allowed us to simultaneously investigate the effects of E2 on attached biofilm mass, biofilm ultrastructure, and QS activity. Upon E2 treatment, six out of 10 investigated CF isolates showed an increase of attached biofilm mass, whereas biofilms from two tested non-CF laboratory strains (PAO1 and ATCC19660) did not. Moreover, FE-SEM and 3D LSM analyses of the E2 responsive CF biofilms revealed ultrastructural remodeling of biofilm structure at different scales with increased formation of prominent biofilm spots, enhanced coverage with extracellular polymeric substance (EPS), and extended average surface roughness. QS activity measurements performed in biofilm supernatants via luminescence acyl homoserine lactone (AHL) reporter assays further showed that E2 treatment may also modulate QS signaling, as shown in an E2 sensitive CF isolate. Together, our results suggest the biofilm modulating effects of E2 on various clinical CF isolates that are documented by both biomass and ultrastructural changes of biofilms. The gained new insight into the influence of steroid hormones on P. aeruginosa biofilm phenotypes might pave the way for novel future approaches in personalized medicine based on the patients' sex and hormonal status.

Antimicrobial Photodynamic Therapy: Latest Developments with a Focus on Combinatory Strategies.

Antimicrobial photodynamic therapy (aPDT) has become a fundamental tool in modern therapeutics, notably due to the expanding versatility of photosensitizers (PSs) and the numerous possibilities to combine aPDT with other antimicrobial treatments to combat localized infections. After revisiting the basic principles of aPDT, this review first highlights the current state of the art of curative or preventive aPDT applications with relevant clinical trials. In addition, the most recent developments in photochemistry and photophysics as well as advanced carrier systems in the context of aPDT are provided, with a focus on the latest generations of efficient and versatile PSs and the progress towards hybrid-multicomponent systems. In particular, deeper insight into combinatory aPDT approaches is afforded, involving non-radiative or other light-based modalities. Selected aPDT perspectives are outlined, pointing out new strategies to target and treat microorganisms. Finally, the review works out the evolution of the conceptually simple PDT methodology towards a much more sophisticated, integrated, and innovative technology as an important element of potent antimicrobial strategies.

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.

Multiplexed detection and differentiation of bacterial enzymes and bacteria by color-encoded sensor hydrogels.

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of α-glucosidase, ß-galactosidase and ß-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns, Staphylococcus aureus strain RN4220, methicillin-resistant S. aureus (MRSA) strain N315, both producing α-glucosidase, but not ß-glucuronidase and ß-galactosidase, Escherichia coli strain DH5α, producing ß-glucuronidase and α-glucosidase, but not ß-galactosidase, and the enterohemorrhagic E. coli (EHEC) strain E32511, producing ß-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.

DNA nuclear targeting sequences for enhanced non-viral gene transfer: An in vitro and in vivo study.

An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NFs are optimized short nucleotide sequences able to interact with the transcription factor nuclear factor κB (NF-κB), which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNAs featuring a common backbone and only varying in their 3NF sequences. These constructions were then transfected under various experimental settings. In vitro, cationic polymer-assisted pDNA delivery in five human-derived cell lines showed the potential advantage of 3NF carrying pDNA in diverse cellular contexts. In vivo, naked pDNAs were hydrodynamically delivered to muscle hindlimbs in healthy mice; this direct accurate comparative (in the absence of any gene carrier) revealed modest but consistent trends in favor of the pDNAs equipped with 3NF. In summary, the results reported emphasize the implications of various parameters on NF-κB-mediated pDNA nuclear import; under specific conditions, 3NF can provide modest to substantial advantages for pDNA gene transfer, in vitro as well as in vivo. This study thus further underscores the potential of optimized nuclear import for more efficient non-viral gene transfer applications.

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.

Temperature-Sensitive Amphiphilic Non-Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection.

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.

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.

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications, hurdles and possible optimizations.

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well-tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non-viral gene transfer following hydrodynamic limb vein injection.

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.

Antibacterial and transfection activities of nebulized formulations incorporating long n-alkyl chain silver N-heterocyclic carbene complexes.

The development of new antibacterial molecules is essential in view of the emergence of pathogenic strains resistant to multiple antibiotics. Among the infectious pathologies, pulmonary infections are particularly difficult to treat due to the complexity of lung anatomy and the presence of natural barriers such as mucus. At present, the aerosol delivery of antibacterial compounds is still poorly employed. Furthermore, the presence of bacteria in lungs negatively affects aerosolized Cystic Fibrosis gene therapy efficiency. A multi-functional formulation (antibacterial and transfection activities) could increase the therapeutic effect. This work reports the synthesis of new N-heterocyclic carbene silver complexes (Ag-NHC) featuring a lipid chain and the evaluation of their antibacterial potency, especially when delivered following aerosolization. When formulated alone in water, these Ag-NHC displayed remarkable antibacterial activities against some Staphyloccocus aureus strains and Pseudomonas aeruginosa clinical strains. Moreover, combined with cationic lipid and DNA (ternary combination), they could be used to deliver therapeutic genes via aerosolization in infected lungs. Altogether, the data reported herein support n-alkyl chain Ag-NHC as a possible alternative to conventional antibiotics for treating respiratory infections and to combat the emergence of multi-resistant bacteria.

Lipid-Based Quaternary Ammonium Sophorolipid Amphiphiles with Antimicrobial and Transfection Activities.

Twelve new quaternary ammonium sophorolipids with long alkyl chains on the nitrogen atom were synthesized starting from oleic and petroselinic acid-based sophorolipids. These novel derivatives were evaluated for their antimicrobial activity against selected Gram-negative and Gram-positive bacteria and their transfection efficacies on three different eukaryotic cell lines in vitro as good activities were demonstrated for previously synthesized derivatives. Self-assembly properties were also evaluated. All compounds proved to possess antimicrobial and transfection properties, and trends could be observed based on the length of the nitrogen substituent and the total length of the sophorolipid tail. Moreover, all long-chain quaternary ammonium sophorolipids form micelles, which proved to be a prerequisite to induce antimicrobial activity and transfection capacity. These results are promising for future healthcare applications of long-chained quaternary ammonium sophorolipids.

Cardiolipin-Based Lipopolyplex Platform for the Delivery of Diverse Nucleic Acids into Gram-Negative Bacteria.

Antibiotic resistance is a growing public health concern. Because only a few novel classes of antibiotics have been developed in the last 40 years, such as the class of oxazolidinones, new antibacterial strategies are urgently needed [1]. Nucleic acid-based antibiotics are a new type of antimicrobials. However, free nucleic acids cannot spontaneously cross the bacterial cell wall and membrane;consequently, their intracellular delivery into bacteria needs to be assisted. Here, we introduce an original lipopolyplex system named liposome polymer nucleic acid (LPN), capable of versatile nucleic acid delivery into bacteria. We characterized LPN formed with significant therapeutic nucleic acids: 11 nt antisense single-stranded (ss) DNA and double-stranded (ds) DNA of 15 and 95 base pairs (bp), 9 kbp plasmid DNA (pDNA), and 1,000 nt ssRNA. All these complexes were efficiently internalized by two different bacterial species, i.e., Escherichiacoli and Pseudomonasaeruginosa, as shown by flow cytometry. Consistent with intracellular delivery, LPN prepared with an antisense oligonucleotide and directed against an essential gene, induced specific and important bacterial growth inhibition likely leading to a bactericidal effect. Our findings indicate that LPN is a versatile platform for efficient delivery of diverse nucleic acids into Gram-negative bacteria.

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.

Polynucleotide transport through lipid membrane in the presence of starburst cyclodextrin-based poly(ethylene glycol)s.

Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.

Ruthenium(II) Polypyridyl Complexes as Photosensitizers for Antibacterial Photodynamic Therapy: A Structure-Activity Study on Clinical Bacterial Strains.

As a growing public health concern, the worldwide spread of antimicrobial resistance urges the development of new therapies. Antibacterial photodynamic therapy (a-PDT) may be an alternative to conventional antibiotic therapy. Herein we report the synthesis and characterization of seven original reactive oxygen species (ROS)-producing ruthenium(II) polypyridyl complexes. These are part of a collection of 17 derivatives varying in terms of the nature of the substituent(s), molecular symmetry, electrical charge, and counterions. They were characterized by considering 1) their physical properties (absorption coefficient at irradiation wavelength, 1 O2 generation quantum yield, luminescence) and 2) their antibacterial activity in a series of photodynamic assays using Gram-positive and Gram-negative bacteria of clinical relevance. The results unveiled some structure-activity relationships: one derivative that combines multiple beneficial features for a-PDT was effective against all the bacteria considered, regardless of their Gram status, species, or antibiotic resistance profile. This systematic study could guide the design of next-generation ruthenium-based complexes for enhanced antibacterial photodynamic strategies.