NANoREG Characterization, solubility and potential toxicity of nanomaterials

Transcription

1 NANoREG Characterization, solubility and potential toicity of nanomaterials Jean-Pascal Piret Julie Laloy Jorge Mejia Namur Nanosafety Center (NNC) University of Namur Department of Pharmacy

2 Namur Nanosafety Centre (NNC) Multidisciplinary platform Coordination Coordinator Dr Olivier Toussaint (URBC) Characterization (PMR-LARN) Prof. Stéphane Lucas Dr Omar Lozano Dr Jorge Mejia Mendoza In Vitro Toicology (URBC) Dr Jean-Pascal Piret Elise Dumortier In Vivo Toicology (Dpt. Of Pharmacy) Prof. Jean-Michel Dogné Dr Julie Laloy Lutfiye Alpan

3 WP2 Synthesis, supplying and characterization Tasks in NANoREG Task 2.3: NPs characterization SOPs for regulatory purposes In this task, all procedures for establishment the physicochemical information requested by REACH will be evaluated and revisions validated Task 2.4 Test item preparation, eposure, dose and fate for regulatory purposes and toicology This task will include development, evaluation and validation of 1) suitable NPs test item dispersion protocols for ecotoicity, in vitro cell- and in vivo instillation studies as well as air-way eposure to test animals; 2) modelling and measurement procedures for accurate quantification of eposure concentrations and characteristics, including NPs fate in the different tests mediums; 3) modelling and measurement procedures for accurate quantification of direct NPs dose to organ, cell or sub-compartment WP5 Advancement of Regulatory Risk Assessment and Testing Task 5.2 Development of solubility testing procedures This task will evaluate the applicability of solubility testing, as used in drug development, and review similar results achieved in other projects, with a view to developing a solubility testing procedure to be applied in rapid NPs screening Task 5.3 The relevance of barriers This task will evaluate the capability of the various NPs proposed in this project to cross five biological barriers (i.e. blood-brain barrier, skin, intestine, lung and oral mucosa) using alternative in vitro models. Several subtasks: intestinal model, airway model, mucosa barrier model, in vitro Blood Brain Barrier model, biomimetic lipid membranes Task 5.5: In vitro toicity assays connected to regulatory questions Identify and optimize the most suitable in vitro methodology to evaluate the toicity for the relevant route toicological endpoint.

4 List of selected NPs SiO 2 NPs NM200 TiO 2 NPs NM101 ZnO NPs NM110 Powders Fluorescent 50 nm positively and negatively charged SiO 2 NPs (task 5.3) Ag NPs (NM300K; spherical NPs 15 nm) Ag NPs (NM302; elongated rods; thickness of nm, length 5-10 µm) Suspensions

5 Task 2.3: NPs characterization SOPs for regulatory purposes Writing of a SOP for NPs size determination with the Centifugal Liquid Sedimentation (CLS) submitted for revision to the work package leader last November 2014 The UNamur is the only NanoReg partner working with the CLS technique instead of the DLS technique.. - Size distribution of particles in liquid sample - Agglomeration degree of nanoparticles - Estimation of number and mass of particles in liquid sample (Proposed as reference technique in revised version of TG 110 granulometry (Deliverable 2.3)

6 Task 2.4: Test item preparation, eposure, dose and fate for regulatory purposes and toicology NPs dispersion procedure Use of a common dispersion protocol (Nanogenoto) SOP for probe-sonicator calibration of delivered acoustic power and de-agglomeration efficiency use of recommended Branson Sonifier S-450D Sonicator (400 Watt and frequency 20 khz) with a 13 mm probe diameter; Selected the lowest sonicator output setting (10% amplitude). Operate it in continuous mode Record the water temperature increase for the initial 5 minutes with a time-resolution of no more than 30 seconds If your sonicator is unable to give the estimated amplitude, please enter the P(ac) for nearest possible amplitude to estimate duration of sonication Your P(ac): 9,7 Total delivered acoustic energy (J): 7056 Your estimated time of sonication: 725, sec. The J corresponds to the total delivered acoustic energy in the NANOGENOTOX dispersion protocols 12 min 20 sec Analysis of size distribution of SiO 2 NPs NM200

7 A Task 2.4: NANoREG requirements Characterization of NPs: Behaviour of NPs in cell culture media (agglomeration, solubilisation, etc ) Analysis of NPs morphology by TEM (stock solution, in culture medium) Analysis of NPs size distribution (DLS, CLS, etc ) in stock solution, in cell culture media (impact of the solvant) After different incubation times -0h, 24h- (effect of incubation times) Mandatory principles: Preparation of a stock NPs solution at 2.56 mg/ml in water % BSA Sonication following the Nanogenoto protocol Dilution of NPs in culture medium for in vitro assays NPs powder for single use only NPs stock solution for single use only

8 Task 2.4: ZnO (NM110) NPs morphology and size distribution Morphology (TEM) and size distribution (CLS) analysis of ZnO NPs (NM110) in water stock solution and after dilution in different cell culture media

9 Task 5.2 Development A of solubility testing procedures 1) Writing of a review on the techniques available to assess the solubility of NPs in collaboration with the other partners of WP5.2 Description of atomic absoprtion spectroscopy (AAS), HPLC and ultracentrifugation techniques (Nanotoicology, in revision) 2) Analysis of NPs solubilisation after different incubation times in several media by Detection of + ions-tetra-(4-aminophenyl) porphyrine (T4APP) complees after ultracentrifugation (100,000g, sedimentation of NPs) or AAS coupled to ultracentifugation (100,000g, sedimentation of NPs)

10 A Lead: UNamur ; Co-lead : ISS Task 5.3 in vitro barrier models Participating partners: LEITAT, UAB, ANSES, CNR, GAIKER, TCD, UiB, Chalmers Establishment of biological barriers models (Unamur intestinal barrier) Subtask : Intestinal barrier (Five partners) Subtask : Airway epithelium (Three partners) Subtask : Oral mucosa (One partner) Subtask : BBB (2 partners) Subtask : Skin (one partner) Subtask 5.3.6: Biomimetic lipid membranes (One partner) Evaluation of the potential crossing of NPs trought different biological barriers (different methods) NPs Culture medium + NPs (membrane 1 µm pore size) Basolateral medium Evaluation of the potential toicity of NPs NPs concentrations used for crossing assay

11 A NPs crossing: detection methods Writing of a SOP for evaluation of NPs crossing (in collaboration with the co-leader ISS) Detection methods for the evaluation of NPs crossing Partners Methods - Instruments NP uptake/internalisation Crossing ISS TCD FUNDP CHALMER S ANSES LEITAT SEM/EDX Fluorescence microscopy* TEM Confocal microscope TEM Confocal microscope Particle accelerator for PIXE analysis Fluorometer* Quartz crystal microbalance TEM Tof-SIMS Fluorometer* Fluorescence microscopy* ICP-MS HPLC-MS/MS (sub contract) (collaboration with BfR partner) CNR To be update GAIKER TEM UAB UiB * For fluorescent NP TEM Cytometer TEM CytoViva Ultra High Resolution Imaging (URI) NP binding to surfaces and interfaces

12 Fluorescent 50 nm positively and negatively charged NPs Fluorescent SiO2 NPs common to all partners as starting NPs to evaluate the accuracy of crossing SOP Evaluate the potential crossing of these fluorescent SiO2 NPs throught the different biological barrier models Analysis of NPs penetration inside biological barrier by confocal microscopy or fluorescent microscopy Des Rieu et al. 2005

13 Evaluation of potential toicity of NPs on Caco-2 cells Evaluation of NPs toicity to chose subtoic NPs concentrations MTS assay SOP 96 well plate NPs concentrations: 100, 50, 25, 10 and 1 µg/ml of NPs Use a common positive control (to be sure that cells are able to die "correctly" and similarly in all labs)

14 Evaluation of potential toicity of sonicated or stirred NPs on different cell lines NPs sonicated (Nanogenoto protocol) stirred Same dispersion time same solvant (water + BSA) Comparison of NPs toicological effects on different cell lines

15 Task 5.5 In vitro toicity assays connected to regulatory questions 1) Participation to interlaboratory studies: 1.1) Cytotoicity (MTS assay) THP-1 cells (immune cells) SiO2 NPs (NM200) ZnO NPs (NM110) Ag NPs (NM300K) 1.2) Inflammation (using common protocols) Ag NPs (NM302) 2) Genotoicity (labelling of phosphorylated histone H2AX) CTL Etoposide

16 Conclusions All tasks are on their way (publications, SOPs, deliverables, etc ) Cross link between WP2 (Deliverable 2.3 and 2.4) and WP5 No impact of culture medium or incubation time on NPs size distribution (NM110) No apparent difference of NPs size distribution in different culture media Problem: Solubilisation of fluorescent NPs Different toic effect depending on dispersion method and cell type Agglomeration state (Sonicated NPs > stirred NPs) Alteration of the NPs surface Different solubilisation