Pseudobacterial nanocarriers for intracellular delivery of anti-infectives

Difficulties in the access and effective treatment of sheltered intracellular bacterial infections may potentially be overcome by encapsulating drugs into delivery systems functionalised with bacteria-derived invasive proteins. Although the potential of these proteins is clear, the promising application and indeed characteristics of invasive particles remains to be fully explored. The objectives of this study were therefore to determine the influence of shape on bacteriomimetic system characteristics, using invasive spherical and aspherical polymeric nanocarriers. Aspherical nanoparticles were prepared by an optimised combination of chemical functionalisation and thermomechanical stretching. InvA497, a C-terminal fragment of the Yersinia-derived protein invasin, was covalently coupled onto the surface of both spherical and aspherical nanoparticles. In vitro studies using drug-free nanoparticles indicated shape-dependent differences on receptor-mediated uptake by epithelial cells, being slightly faster for spherical nanoparticles. Both types of nanoparticles were then loaded with a preparation of antibiotic gentamicin, and tested for their ability to kill intracellular Shigella flexneri in human epithelial cells. Aspherical systems led to a higher killing of intracellular bacteria, potentially due to a more favorable drug release profile. This study provides a proof of concept that InvA497-functionalised aspherical bacteriomimetic nanocarriers may efficiently deliver otherwise non-permeable antibiotics across host cell membranes, enabling effective treatment of intracellular infections.Eine wirksame Behandlung von intrazellulärer lokalisierten und auf diese Weise geschützten bakteriellen Infektionen kann mit Hilfe von spezieller nanopartikulären Systemen, die mit invasiven, von Bakterien abgeleiteten Proteinen funktionalisiert sind, erreicht werden. Obwohl das Potenzial dieser Proteinen bereits bekannt ist, sind vielversprechende Anwendungen und Eigenschaften dieser bakteriomimetischen Transportsysteme noch gründlich zu erforschen. Ziel dieser Arbeit war es, den Einfluss von zwei unterschiedlichen Partikelformen (sphärisch und asphärisch) auf die Wirksamkeit dieser bakteriomimetischen Systeme zu untersuchen. Asphärische Nanopartikel wurden durch eine optimale Kombination von chemischer Funktionalisierung und thermomechanischer Dehnung vorbereitet. InvA497, ein C-Endfragment des invasiven, von Yersinia abgeleiteten Proteins «Invasin», wurde kovalent an die Nanopartikeloberfläche gekoppelt. In vitro Untersuchungen mit wirkstofffreien Nanopartikeln zeigten formabhängige Unterschiede in der Rezeptor-vermittelten Aufnahme bei Epithelzellen Hep-2. Bei sphärischen Nanopartikeln war die Aufnahme etwas schneller. Beide Nanopartikelsysteme wurden daraufhin mit dem Antibiotikum Gentamicin geladen und auf deren Fähigkeit geprüft, intrazelluläre Shigella flexneri Erreger in menschlichen Epithelzellen zu töten. Asphärische Systeme zeigten eine höhere Fähigkeit zur Abtötung der intrazellulären Bakterien, möglicherweise bedingt durch ein vorteilhaftes Wirkstofffreisetzungsprofil. Mit dieser Untersuchung konnte nachgewiesen werden, dass InvA497-funktionalisierte, asphärische, bakteriomimetische Nanoträger nicht permeable Antibiotika durch die Wirtszellmembran effizient transportieren können, um intrazelluläre Infektionen behandeln zu können

Atrophic pseudarthrosis of humeral diaphyseal fractures: medico-legal implications and methodological analysis of the evaluation

Humeral shaft fractures account for 1- 3% of all fractures and about 20-27% of those involving the humerus. In the past they were often conservatively treated, with an acceptable consolidation rate. Open reduction and internal fixation (ORIF) is the best choice in polytrauma patients, in complex or pathological fractures and in those associated with vascular injuries. Regardless the type of fixation used, these fractures can evolve into delayed union or pseudarthrosis (PSA). It should be noted that the humeral shaft itself has a high intrinsic healing potential, due to the blood supply provided by the surrounding muscles. The aim of this work is to evaluate whether the causes that led to the development of atrophic pseudarthrosis in a humeral diaphyseal fracture are attributable to inadequate management of this fearful complication and to highlight the possible medico-legal repercussions. We will try to verify whether the currently used forensic evaluation parameters of permanent disability are appropriate and adequate in relation to the complexity of such injuries. This complexity also includes the repercussions on the ergonomic efficiency of the entire limb, the relative possible postural alterations, the inevitable extension of the period of traumatic illness and the relative repercussions on the overall compromised structure of the subject

Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections.

The influence of vitamin D3 and its metabolites calcifediol (25(OH)D) and calcitriol on immune regulation and inflammation is well described, and raises the question of potential benefit against bacterial infections. In the current study, 25(OH)D was encapsulated in liposomes to enable aerosolisation, and tested for the ability to prevent pulmonary infection by Pseudomonas aeruginosa. Prepared 25(OH)D-loaded liposomes were nanosized and monodisperse, with a negative surface charge and a 25(OH)D entrapment efficiency of approximately 23%. Jet nebulisation of liposomes was seen to yield an aerosol suitable for tracheo-bronchial deposition. Interestingly, 25(OH)D in either liposomes or ethanolic solution had no effect on the release of the proinflammatory cytokine KC from Pseudomonas-infected murine epithelial cells (LA-4); treatment of infected, human bronchial 16-HBE cells with 25(OH)D liposomes however resulted in a significant reduction in bacterial survival. Together with the importance of selecting an application-appropriate in vitro model, the current study illustrates the feasibility and practicality of employing liposomes as a means to achieve 25(OH)D lung deposition. 25(OH)D-loaded liposomes further demonstrated promising effects regarding prevention of Pseudomonas infection in human bronchial epithelial cells

Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.

The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections. Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri. InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration. InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy

Aspherical and spherical InvA497-functionalized nanocarriers for intracellular delivery of anti-infective agents

The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections. Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri. InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration. InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy

Peer review of the pesticide risk assessment for the active substance thiabendazole in light of confirmatory data submitted

Abstract The conclusions of the EFSA following the peer review of the initial risk assessment carried out by the competent authority of the rapporteur Member State, Spain, for the pesticide active substance thiabendazole are reported. The context of the peer review was that requested by the European Commission following the submission and evaluation of confirmatory information with regard to the endocrine disruption potential of the substance. The conclusions were reached on the basis of the evaluation of the representative uses of thiabendazole as a fungicide on seed potato, apple and pear and citrus. Assessments not finalised together with the missing information identified as being required by the mandate are listed. Concerns are identified

Targeted review of maximum residue levels (MRLs) for fenarimol

Abstract In accordance with Article 43 of Regulation (EC) 396/2005, EFSA received a request from the European Commission to review the existing maximum residue levels (MRLs) for the non‐approved active substance fenarimol in view of the possible lowering of the MRLs. EFSA investigated the origin of the current EU MRLs. For existing EU MRLs that reflect previously authorised uses in the EU, or that are based on obsolete Codex maximum residue limits, or import tolerances that are not required any longer, EFSA proposed the lowering to the limit of quantification. EFSA performed a chronic and acute dietary risk assessment for the revised list of MRLs to allow risk managers to take the appropriate decisions. For some commodities, further risk management discussions are required to decide which of the risk management options proposed by EFSA should be implemented in the EU MRL legislation