• Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors.

      Nafee, Noha; Husari, Ayman; Maurer, Christine K; Lu, Cenbin; de Rossi, Chiara; Steinbach, Anke; Hartmann, Rolf W; Lehr, Claus-Michael; Schneider, Marc (2014-10-28)
      Cystic fibrosis (CF) is a genetic disease mainly manifested in the respiratory tract. Pseudomonas aeruginosa (P. aeruginosa) is the most common pathogen identified in cultures of the CF airways, however, its eradication with antibiotics remains challenging as it grows in biofilms that counterwork human immune response and dramatically decrease susceptibility to antibiotics. P. aeruginosa regulates pathogenicity via a cell-to-cell communication system known as quorum sensing (QS) involving the virulence factor (pyocyanin), thus representing an attractive target for coping with bacterial pathogenicity. The first in vivo potent QS inhibitor (QSI) was recently developed. Nevertheless, its lipophilic nature might hamper its penetration of non-cellular barriers such as mucus and bacterial biofilms, which limits its biomedical application. Successful anti-infective inhalation therapy necessitates proper design of a biodegradable nanocarrier allowing: 1) high loading and prolonged release, 2) mucus penetration, 3) effective pulmonary delivery, and 4) maintenance of the anti-virulence activity of the QSI. In this context, various pharmaceutical lipids were used to prepare ultra-small solid lipid nanoparticles (us-SLNs) by hot melt homogenization. Plain and QSI-loaded SLNs were characterized in terms of colloidal properties, drug loading, in vitro release and acute toxicity on Calu-3 cells. Mucus penetration was studied using a newly-developed confocal microscopy technique based on 3D-time-lapse imaging. For pulmonary application, nebulization efficiency of SLNs and lung deposition using next generation impactor (NGI) were performed. The anti-virulence efficacy was investigated by pyocyanin formation in P. aeruginosa cultures. Ultra-small SLNs (<100nm diameter) provided high encapsulation efficiency (68-95%) according to SLN composition, high burst in phosphate buffer saline compared to prolonged release of the payload over >8h in simulated lung fluid with minor burst. All types and concentrations of plain and QSI-loaded SLNs maintained the viability of Calu-3 cells. 3D time-lapse confocal imaging proved the ability of SLNs to penetrate into artificial sputum model. SLNs were efficiently nebulized; NGI experiments revealed their deposition in the bronchial region. Overall, nanoencapsulated QSI showed up to sevenfold superior anti-virulence activity to the free compound. Most interestingly, the plain SLNs exhibited anti-virulence properties themselves, which was shown to be related to anti-virulence effects of the emulsifiers used. These startling findings represent a new perspective of ultimate significance in the area of nano-based delivery of novel anti-infectives.
    • Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

      Frank, Julia; Richter, Maximilian; de Rossi, Chiara; Lehr, Claus-Michael; Fuhrmann, Kathrin; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-08-17)
      Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including -80 °C, 4 °C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, β-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.
    • Itaconic Acid Increases the Efficacy of Tobramycin against Biofilms.

      Ho, Duy-Khiet; de Rossi, Chiara; Loretz, Brigitta; Murgia, Xabier; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-07-22)
      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.
    • Micro-rheological properties of lung homogenates correlate with infection severity in a mouse model of Pseudomonas aeruginosa lung infection.

      Murgia, Xabier; Kany, Andreas M; Herr, Christian; Ho, Duy-Khiet; de Rossi, Chiara; Bals, Robert; Lehr, Claus-Michael; Hirsch, Anna K H; Hartmann, Rolf W; Empting, Martin; et al. (Nature publishing group (NPG), 2020-10-05)
      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.
    • PLGA nanocapsules improve the delivery of clarithromycin to kill intracellular Staphylococcus aureus and Mycobacterium abscessus.

      Anversa Dimer, Frantiescoli; de Souza Carvalho-Wodarz, Cristiane; Goes, Adriely; Cirnski, Katarina; Herrmann, Jennifer; Schmitt, Viktoria; Pätzold, Linda; Abed, Nadia; de Rossi, Chiara; Bischoff, Markus; et al. (Elsevier, 2019-11-18)
      Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70-80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.
    • Polysaccharide Submicrocarrier for Improved Pulmonary Delivery of Poorly Soluble Anti-infective Ciprofloxacin: Preparation, Characterization, and Influence of Size on Cellular Uptake.

      Ho, Duy-Khiet; Costa, Ana; de Rossi, Chiara; de Souza Carvalho-Wodarz, Cristiane; Loretz, Brigitta; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2018-02-21)
      The majority of the currently used and developed anti-infectives are poorly water-soluble molecules. The poor solubility might lead to limited bioavailability and pharmacological action of the drug. Novel pharmaceutical materials have thus been designed to solve those problems and improve drug delivery. In this study, we propose a facile method to produce submicrocarriers (sMCs) by electrostatic gelation of anionic ß-cyclodextrin (aß-CD) and chitosan. The average hydrodynamic size ranged from 400 to 900 nm by carefully adjusting polymer concentrations and N/C ratio. The distinct host-guest reaction of cyclodextrin derivative is considered as a good approach to enhance solubility, and prevent drug recrystallization, and thus was used to develop sMC to improve the controlled release profile of a poorly soluble and clinically relevant anti-infective ciprofloxacin. The optimal molar ratio of ciprofloxacin to aß-CD was found to be 1:1, which helped maximize encapsulation efficiency (∼90%) and loading capacity (∼9%) of ciprofloxacin loaded sMCs. Furthermore, to recommend the future application of the developed sMCs, the dependence of cell uptake on sMCs size (500, 700, and 900 nm) was investigated in vitro on dTHP-1 by both flow cytometry and confocal microscopy. The results demonstrate that, regardless of their size, an only comparatively small fraction of the sMCs were taken up by the macrophage-like cells, while most of the carriers were merely adsorbed to the cell surface after 2 h incubation. After continuing the incubation to reach 24 h, the majority of the sMCs were found intracellularly. However, the sMCs had been designed to release sufficient amount of drug within 24 h, and the subsequent phagocytosis of the carrier may be considered as an efficient pathway for its safe degradation and elimination. In summary, the developed sMC is a suitable system with promising perspectives recommended for pulmonary extracellular infection therapeutics.
    • Squalenyl Hydrogen Sulfate Nanoparticles for Simultaneous Delivery of Tobramycin and an Alkylquinolone Quorum Sensing Inhibitor Enable the Eradication of P. aeruginosa Biofilm Infections.

      Ho, Duy-Khiet; Murgia, Xabier; de Rossi, Chiara; Christmann, Rebekka; Hüfner de Mello Martins, Antonio G; Koch, Marcus; Andreas, Anastasia; Herrmann, Jennifer; Müller, Rolf; Empting, Martin; et al. (Wiley, 2020-04-03)
      Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self-assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug-loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16-fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use.
    • Synthesis and Biopharmaceutical Characterization of Amphiphilic Squalenyl Derivative Based Versatile Drug Delivery Platform.

      Ho, Duy-Khiet; Christmann, Rebekka; Murgia, Xabier; de Rossi, Chiara; Frisch, Sarah; Koch, Marcus; Schaefer, Ulrich F; Loretz, Brigitta; Desmaele, Didier; Couvreur, Patrick; et al. (Frontiers, 2020-10-19)
      Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.