The Role of Airway Mucus as a Barrier to Nanoparticles: Implications for Pulmonary Drug Delivery

A layer of mucus covers the surface of the pulmonary airways. Mucus is a hydrogel mainly composed of water, mucins (glycoproteins), DNA, proteins, lipids, and cell debris, which conditions the inhaled air and protects us from airborne threats. The natural protective role of mucus is nowadays acknowledged as a major barrier to be overcome in non-invasive drug delivery. The heterogeneity of mucus components offers a wide range of potential chemical interaction sites for macromolecules, while the mesh-like architecture given to mucus by the intermolecular cross- linking of mucin molecules results in a dense network that physically, and in a size-dependent manner, hinders the diffusion of macromolecules and nanoparticles through mucus. In this thesis, native airway mucus samples of porcine and human origin have been exhaustively characterized utilizing techniques that allow its investigation while preserving its original viscoelastic properties. The rheological properties and the penetration of differently sized nanoparticles either mechanically mixed with mucus or deposited as an aerosol onto mucus layers have been investigated. Additionally, a significant research effort has been spent in the development of in vitro models aimed at mimicking the bronchial region under diverse physiological conditions, including bacterial infection. These models have been proved suitable for investigating the translocation of nanoparticle-based drug delivery systems through the airways as well as for testing the efficacy of antibiotics in the context of cystic fibrosis

Itaconic Acid Increases the Efficacy of Tobramycin against Pseudomonas aeruginosa Biofilms

The search for novel therapeutics against pulmonary infections, in particular Pseudomonas aeruginosa (PA) biofilm infections, has been intense to deal with the emergent rise of antimicrobial resistance. Despite the numerous achievements in drug discovery and delivery strategies, only a limited number of therapeutics reach the clinic. To allow a timely preclinical development, a formulation should be highly effective, safe, and most importantly facile to produce. Thus, a simple combination of known actives that enhances the therapeutic efficacy would be a preferential choice compared to advanced drug delivery systems. In this study, we propose a novel combination of an anti-inflammatory agent—itaconic acid (itaconate, IA)—and an approved antibiotic—tobramycin (Tob) or ciprofloxacin (Cipro). The combination of Tob and IA at a molar ratio of 1:5 increased the biofilm eradicating efficacy in the strain PA14 wild type (wt) by ~4-fold compared to Tob alone. In contrast, such effect was not observed for the combination of IA with Cipro. Subsequent studies on the influence of IA on bacterial growth, pyocyanin production, and Tob biofilm penetration indicated that complexation with IA enhanced the transport of Tob through the biofilm. We recommend the simple and effective combination of Tob:IA for further testing in advanced preclinical models of PA biofilm infections

Early Cerebral Hemodynamic, Metabolic, and Histological Changes in Hypoxic–Ischemic Fetal Lambs during Postnatal Life

The hemodynamic, metabolic, and biochemical changes produced during the transition from fetal to neonatal life may be aggravated if an episode of asphyxia occurs during fetal life. The aim of the study was to examine regional cerebral blood flow (RCBF), histological changes, and cerebral brain metabolism in preterm lambs, and to analyze the role of oxidative stress in the first hours of postnatal life following severe fetal asphyxia. Eighteen chronically instrumented newborn lambs were randomly assigned to either a control group or the hypoxic–ischemic (HI) group, in which case fetal asphyxia was induced just before delivery. All the animals were maintained on intermittent positive pressure ventilation for 3 h after delivery. During the HI insult, the injured group developed acidosis, hypoxia, hypercapnia, lactic acidosis, and tachycardia (relative to the control group), without hypotension. The intermittent positive pressure ventilation transiently improved gas exchange and cardiovascular parameters. After HI injury and during ventilatory support, there continued to be an increased RCBF in inner regions among the HI group, but no significant differences were detected in cortical flow compared to the control group. Also, the magnitude of the increase in TUNEL positive cells (apoptosis) and antioxidant enzymes, and decrease of ATP reserves was significantly greater in the brain regions where the RCBF was not higher. In conclusion, our findings identify early metabolic, histological, and hemodynamic changes involved in brain damage in premature asphyxiated lambs. Such changes have been described in human neonates, so our model could be useful to test the safety and the effectiveness of different neuroprotective or ventilation strategies applied in the first hours after fetal HI injury

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Vukosavljevic B, Hittinger M, Hachmeister H, et al. Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells. Journal of Biophotonics. 2019;12(6): e201800052

Physiological, biochemical, and biophysical characterization of the lung-lavaged spontaneously-breathing rabbit as a model for respiratory distress syndrome

Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in spontaneously-breathing premature infants with respiratory distress syndrome (RDS). Surfactant administration techniques compatible with nCPAP ventilation strategy are actively investigated. Our aim is to set up and validate a respiratory distress animal model that can be managed on nCPAP suitable for surfactant administration techniques studies. Surfactant depletion was induced by bronchoalveolar lavages (BALs) on 18 adult rabbits. Full depletion was assessed by surfactant component analysis on the BALs samples. Animals were randomized into two groups: Control group (nCPAP only) and InSurE group, consisting of a bolus of surfactant (Poractant alfa, 200 mg/kg) followed by nCPAP. Arterial blood gases were monitored until animal sacrifice, 3 hours post treatment. Lung mechanics were evaluated just before and after BALs, at the time of treatment, and at the end of the procedure. Surfactant phospholipids and protein analysis as well as surface tension measurements on sequential BALs confirmed the efficacy of the surfactant depletion procedure. The InSurE group showed a significant improvement of blood oxygenation and lung mechanics. On the contrary, no signs of recovery were appreciated in animals treated with just nCPAP. The surfactant-depleted adult rabbit RDS model proved to be a valuable and efficient preclinical tool for mimicking the clinical scenario of preterm infants affected by mild/moderate RDS who spontaneously breathe and do not require mechanical ventilation. This population is of particular interest as potential target for the non-invasive administration of surfactant

Micro-rheological properties of lung homogenates correlate with infection severity in a mouse model of Pseudomonas aeruginosa lung infection

Lung infections caused by Pseudomonas aeruginosa pose a serious threat to patients suffering from, among others, cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis, often leading to life-threatening complications. The establishment of a chronic infection is substantially related to communication between bacteria via quorum-sensing networks. In this study, we aimed to assess the role of quorum-sensing signaling molecules of the Pseudomonas quinolone signal (PQS) and to investigate the viscoelastic properties of lung tissue homogenates of PA-infected mice in a prolonged acute murine infection model. Therefore, a murine infection model was successfully established via intra-tracheal infection with alginate-supplemented Pseudomonas aeruginosa NH57388A. Rheological properties of lung homogenates were analyzed with multiple particle tracking (MPT) and quorum-sensing molecules were quantified with LC–MS/MS. Statistical analysis of bacterial load and quorum-sensing molecules showed a strong correlation between these biomarkers in infected lungs. This was accompanied by noticeable changes in the consistency of lung homogenates with increasing infection severity. Furthermore, viscoelastic properties of the lung homogenates strongly correlated with bacterial load and quorum sensing molecules. Considering the strong correlation between the viscoelasticity of lung homogenates and the aforementioned biomarkers, the viscoelastic properties of infected lungs might serve as reliable new biomarker for the evaluation of the severity of P. aeruginosa infections in murine models

Surfactant-assisted distal pulmonary distribution of Budesonide revealed by mass spectrometry imaging

13openInternationalBothDirect lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle (n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa (n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs (n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lungopenZecchi, Riccardo; Franceschi, Pietro; Tigli, Laura; Pioselli, Barbara; Mileo, Valentina; Murgia, Xabier; Salomone, Fabrizio; Pieraccini, Giuseppe; Usada, Haruo; Schmidt, Augusto F; Hillman, Noah H.; Kemp, Matthew W.; Jobe, Alan H.Zecchi, R.; Franceschi, P.; Tigli, L.; Pioselli, B.; Mileo, V.; Murgia, X.; Salomone, F.; Pieraccini, G.; Usada, H.; Schmidt, A.F.; Hillman, N.H.; Kemp, M.W.; Jobe, A.H

A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development

A Compartment-Based Mathematical Model for Studying Convective Aerosol Transport in Newborns Receiving Nebulized Drugs during Noninvasive Respiratory Support

Nebulization could be a valuable solution to administer drugs to neonates receiving noninvasive respiratory support. Small and irregular tidal volumes and air leaks at the patient interface, which are specific characteristics of this patient population and are primarily responsible for the low doses delivered to the lung (DDL) found in this application, have not been thoroughly addressed in in vitro and in vivo studies for quantifying DDL. Therefore, we propose a compartment-based mathematical model able to describe convective aerosol transport mechanisms to complement the existing deposition models. Our model encompasses a mechanical ventilator, a nebulizer, and the patient; the model considers the gas flowing between compartments, including air leaks at the patient–ventilator interface. Aerosol particles are suspended in the gas flow and homogeneously distributed. The impact of breathing pattern variability, volume of the nebulizer, and leaks level on DDL is assessed in representative conditions. The main finding of this study is that convective mechanisms associated to air leaks and breathing patterns with tidal volumes smaller than the nebulizer dramatically reduce the DDL (up to 70%). This study provides a possible explanation to the inconsistent results of drug aerosolization in clinical studies and may provide guidance to improve nebulizer design and clinical procedures

The role of mucus on drug transport and its potential to affect therapeutic outcomes.

A layer of mucus covers the surface of all wet epithelia throughout the human body. Mucus is a hydrogel mainly composed of water, mucins (glycoproteins), DNA, proteins, lipids, and cell debris. This complex composition yields a tenacious viscoelastic hydrogel that lubricates and protects the exposed epithelia from external threats and enzymatic degradation. The natural protective role of mucus is nowadays acknowledged as a major barrier to be overcome in non-invasive drug delivery. The heterogeneity of mucus components offers a wide range of potential chemical interaction sites for macromolecules, while the mesh-like architecture given to mucus by the intermolecular cross-linking of mucin molecules results in a dense network that physically, and in a size-dependent manner, hinders the diffusion of nanoparticles through mucus. Consequently, drug diffusion, epithelial absorption, drug bioavailability, and ultimately therapeutic outcomes of mucosal drug delivery can be attenuate