Combining Cytotoxicity Assessment and Xenopus laevis Phenotypic Abnormality Assay as a Predictor of Nanomaterial Safety

Karamallah Al‐Yousuf1, Carl A. Webster2, Grant N. Wheeler2, Francesca Baldelli Bombelli3, Victoria Sherwood1

1 Skin Tumour Laboratory, Jacqui Wood Cancer Centre, Division of Cancer Research, School of Medicine, University of Dundee, Dundee, 2 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, 3 Department of Chemistry, Materials and Chemical Engineering “G.Natta,” Politecnico di Milano, Milano
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.13
DOI:  10.1002/cptx.25
Online Posting Date:  August, 2017
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Abstract

The African clawed frog, Xenopus laevis, has been used as an efficient pre‐clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high‐throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever‐increasing volume of biomedical nanoparticles (NPs) in development, their unique physico‐chemical properties challenge the use of standard toxicology assays. Here, we present a protocol that directly compares the sensitivity of X. laevis development as a tool to assess potential NP toxicity by observation of embryo phenotypic abnormalities/lethality after NP exposure, to in vitro cytotoxicity obtained using mammalian cell lines. In combination with conventional cytotoxicity assays, the X. laevis phenotypic assay provides accurate data to efficiently assess the safety of novel biomedical NPs. © 2017 by John Wiley & Sons, Inc.

Keywords: nanoparticles; nanotoxicity; physical‐chemical characterization of nanoparticles; cytotoxicity; Xenopus laevis embryos

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Physicochemical Characterization of Nanoparticles (NPs)
  • Support Protocol 1: Transmission Electron Microscopy for Nanoparticle Characterization
  • Basic Protocol 2: Cytotoxicity Assessment of Nanoparticle Treatment
  • Support Protocol 2: Trypan Blue Exclusion Assay
  • Support Protocol 3: Immunoblotting for Apoptotic Markers
  • Basic Protocol 3: X. Laevis Phenotypic Abnormality Assay for Nanotoxicity Assessment
  • Support Protocol 4: Harvesting X. Laevis Embryos
  • Support Protocol 5: Whole‐Mount Imaging of X. Laevis Embryos for Fluorescent Nanoparticle Uptake
  • Alternate Protocol 1: TEM Imaging of X. Laevis Embryo Sections for Nanoparticle Uptake
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Physicochemical Characterization of Nanoparticles (NPs)

  Materials
  • Nanoparticle (NP) stock dispersions (concentrations and nanomaterials tested are to be pre‐determined by the experimenter)
  • PBS (see recipe)
  • Growth medium (GM) for mammalian cells, containing supplements (e.g., FBS, amino acids, antibiotics, as required depending on chosen cell types; GM details for specific lines are provided by the supplier or in the scientific literature; all reagents must be cell culture grade)
  • 0.1× Marc's Modified Ringer's (MMR) solution (see recipe)
  • Disposable dynamic light scattering (DLS) cuvettes
  • Dynamic light scattering apparatus

Support Protocol 1: Transmission Electron Microscopy for Nanoparticle Characterization

  Materials
  • Nanoparticle (NP) stock dispersions (concentrations and nanomaterials tested are to be pre‐determined by the experimenter)
  • Transmission electron microscopy (TEM) grid (chosen material depends on NP material and specifics of apparatus and manufacturer)
  • Transmission electron microscopy (TEM) instrument with imaging modality

Basic Protocol 2: Cytotoxicity Assessment of Nanoparticle Treatment

  Materials
  • Mammalian cell lines of choice (use a minimum of 3)
  • Growth medium (GM) for mammalian cells containing supplements (e.g., FBS, amino acids, antibiotics, as required depending on chosen cell types; GM details for specific lines are provided by the supplier or in the scientific literature; all reagents must be cell culture grade)
  • 70% ethanol (Sigma‐Aldrich)
  • 0.05% (w/v) trypsin‐EDTA solution (cell culture grade; Sigma‐Aldrich)
  • Mycoplasma testing kit (we use EZ‐PCR mycoplasma test kit; Gene Flow)
  • Nanoparticle (NP) exposure solution (concentrations and nanomaterials tested are to be pre‐determined by the experimenter)
  • 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) solution (Sigma‐Aldrich; prepared according to the manufacturer's instructions)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich)
  • Sørensen's buffer (see recipe)
  • PBS (see recipe)
  • Liquid N 2 cryogenic cell storage Dewar flask (for long‐term storage of cell stocks; Cole‐Palmer)
  • Water bath (set to 37°C; Thermo Fisher Scientific; add an anti‐microbial agent to the water tray to limit contamination)
  • Class II biological safety cabinet (Monmouth Scientific)
  • Sterile, disposable cell culture plastic ware (e.g., flasks, plates, tubes, tips; for adherent cells, flasks and plates must be cell culture grade)
  • Humidified 37°C, 5% CO 2 cell culture incubator (New Brunswick; add an anti‐microbial agent to the water tray to limit contamination)
  • Inverted light microscope (Olympus)
  • Swing‐out (bucket) centrifuge (Eppendorf)
  • Automated cell counter (e.g., Bio‐Rad TC20) or Neubauer hemocytometer (Merck Millipore)
  • Multichannel pipet (Thermo Fisher Scientific)
  • Microplate spectrophotometer reader (SpectraMax)

Support Protocol 2: Trypan Blue Exclusion Assay

  Additional Materials (also see protocol 1)
  • Mammalian cell lines of choice (use a minimum of 3; see protocol 1 for a detailed list of equipment and reagents required for growing mammalian cell lines)
  • Nanoparticle (NP) exposure solution (concentrations and nanomaterials tested are to be pre‐determined by the experimenter)
  • 0.05% (w/v) trypsin‐EDTA solution (cell culture grade; Sigma‐Aldrich)
  • 0.4% trypan blue solution (Sigma‐Aldrich)

Support Protocol 3: Immunoblotting for Apoptotic Markers

  Additional Materials (also see protocol 1)
  • Mammalian cell lines of choice (use a minimum of 3; see protocol 1 for a detailed list of equipment and reagents required for growing mammalian cell lines)
  • Nanoparticle (NP) exposure solution (concentrations and nanomaterials tested are to be pre‐determined by the experimenter)
  • Cisplatin or other cytotoxic agent (used as a positive control in cell lines of choice; agent and dose should be pre‐determined for each cell line selected)
  • PBS (0.1 to 0.5 liter, cooled to 4°C; see recipe)
  • Lysis buffer (containing protease inhibitors, cooled to 4°C; see recipe)
  • Pierce BCA Protein Assay kit (Thermo Fisher Scientific)
  • Dithiothreitol (DTT; Sigma‐Aldrich)
  • SDS (Sigma‐Aldrich)
  • 4× loading buffer (see recipe)
  • Tris·Cl buffers (pH 8.8 and pH 6.8; see recipes)
  • 40% acrylamide/bisacrylamide (Sigma‐Aldrich)
  • Ammonium persulfate (APS; Sigma‐Aldrich)
  • Tetramethylethylenediamine (TEMED), >99.5% (Sigma‐Aldrich)
  • 10× running buffer (see recipe)
  • 10× transfer buffer (see recipe)
  • Tris‐buffered saline/Tween 20 (TBST; see recipe)
  • Ponceau S solution (see recipe)
  • ECL Western blotting detection reagent (GE Healthcare)
  • Blocking solution (see recipe)
  • Methanol
  • Mouse anti‐PARP1 antibody (F‐2; Santa Cruz Biotechnology, cat. no. SC‐8007)
  • Mouse anti‐α‐tubulin (DM1A; Cell Signalling Technology, cat. no. 3873)
  • Anti‐mouse Horseradish‐peroxidase (HRP)‐conjugated secondary antibody (Cell Signaling Technology, cat. no. 7076)
  • Plastic cell scrapers (Thermo Fisher Scientific)
  • 1.5‐ml Eppendorf microcentrifuge tubes (Thermo Fisher Scientific)
  • Sonicator (e.g., Diagenode Bioruptor Pico Ultrasonicator; Thermo Fisher Scientific)
  • −20°C freezer
  • UV‐Vis Spectrophotometer (Orion AquaMate 8000; Thermo Fisher Scientific)
  • Dry block heating system
  • Mini‐gel tank and associated casting plates, combs, and related items (Mini‐PROTEAN Tetra Vertical Electrophoresis Cell; Bio‐Rad)
  • Protein molecular weight standards (range: 6500 to 205,000 Da; Thermo Fisher Scientific)
  • Gel‐loading tips (range: 0.5 to 200 µl; Thermo Fisher Scientific)
  • Universal power supply (PowerPac; Bio‐Rad)
  • Polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific) or nitrocellulose membrane (Thermo Fisher Scientific)
  • Bent‐tip stainless‐steel forceps (Thermo Fisher Scientific)
  • Sponge pads and filter paper (for blotting; Invitrogen)
  • Shaker plate/roller
  • ChemiDoc XRS+ system (Bio‐Rad)
  • Image analysis software (ChemiDoc Touch, Bio‐Rad)

Basic Protocol 3: X. Laevis Phenotypic Abnormality Assay for Nanotoxicity Assessment

  Materials
  • Nieuwkoop and Faber (NF) stage 1 X. laevis embryos (see protocol 7)
  • 0.1× Marc's Modified Ringer's (MMR) solution (see recipe)
  • Nanoparticle (NP) exposure solution (concentrations and nanomaterials tested are to be pre‐determined by the experimenter)
  • Ethyl 3‐aminobenzoate methanesulfonate (0.6 mg/ml; see recipe)
  • MEMFA fixative (see recipe)
  • PBS (see recipe)
  • PBS/Tween 20 (PBST; see recipe)
  • 2% (w/v) agarose gel (see recipe)
  • Methanol (analytical grade; Sigma‐Aldrich)
  • 25%, 50%, and 75% (v/w) methanol (analytical grade; Sigma‐Aldrich) in PBS
  • Gentamycin (25 µl/ml; optional)
  • Pasteur pipet (we recommend glass; whole embryos are too large to fit into a standard pipet, therefore mark the end with a diamond pen, break off cleanly and fire the end briefly to melt any sharp edges; alternatively, plastic Pasteur pipets can be used with the end removed)
  • 10‐cm2 Petri dish (Thermo Fisher Scientific)
  • Culture incubator (set to desired temperature; see below for details)
  • Stereomicroscope with two‐armed fiber optic illuminator (to allow the angle of illumination to be easily adjusted)
  • Dumont #5 forceps (stainless steel; ultrafine and can be used for carefully manipulating embryos throughout the protocol; Sigma‐Aldrich).
  • 24‐well culture plate (non‐cell culture grade; Thermo Fisher Scientific)
  • 3‐cm2 Petri dish (Thermo Fisher Scientific)
  • Long‐handled scalpel (10 A blades)
  • Light microscope with charge coupled‐device (CCD) digital camera (for whole‐mount imaging of embryos)
  • Glass vials with screw caps (3.5 ml; SGL)
  • Parafilm M wrapping film (Thermo Fisher Scientific)
  • −20°C freezer

Support Protocol 4: Harvesting X. Laevis Embryos

  Materials
  • Female X. laevis adults (two or more)
  • Pregnant mare serum gonadotrophin (PMSG; Intervet)
  • Human chorionic gonadotrophin (hCG; Intervet)
  • 0.1× Marc's Modified Ringer's (MMR) solution (see recipe)
  • One male X. laevis adult
  • Ethyl 3‐aminobenzoate methanesulfonate (0.6 mg/ml; see recipe)
  • Testes buffer (see recipe)
  • 2% cysteine (w/v) de‐jellying solution (see recipe)
  • 25‐G needle (BD Biosciences) and 1‐ml syringe (Thermo Fisher Scientific)
  • Non‐textured, powder‐free gloves (Thermo Fisher Scientific)
  • Culture incubator (set to 17°C)
  • 10‐cm2 Petri dish (Thermo Fisher Scientific)
  • Surgical equipment including scalpels, forceps, and curved scissors
  • −20°C freezer

Support Protocol 5: Whole‐Mount Imaging of X. Laevis Embryos for Fluorescent Nanoparticle Uptake

  Materials
  • 2% (w/v) agarose gel (Sigma‐Aldrich; see recipe)
  • Tadpole stage X. laevis embryos (from NF stage 38 onwards; see Fig.  ; anesthetized [in 0.06% ethyl 3‐aminobenzoate methanesulfonate: Dilute 0.6 mg/ml solution 1/10 in dH 2O, until movement is inhibited] and pre‐exposed to florescent NPs, see step 5, protocol 6 for details; use 20 nm PS‐COOH NPs as a positive control)
  • PBS (see recipe)
  • 3‐cm2 Petri dish (Thermo Fisher Scientific)
  • Long‐handled scalpel (10 A blades)
  • Glass Pasteur pipet (prepared as described in protocol 6)
  • Dumont #5 forceps (stainless steel; ultrafine and can be used for carefully manipulating embryos throughout the protocol; Sigma‐Aldrich)
  • Fluorescent microscope with CCD digital camera

Alternate Protocol 1: TEM Imaging of X. Laevis Embryo Sections for Nanoparticle Uptake

  Additional Materials (also see protocol 6)
  • Tadpole stage X. laevis embryos (from NF stage 38 onwards; see Fig.  ; anesthetized and pre‐exposed to NPs, see step 5, protocol 6 for details)
  • TEM fixing buffer (see recipe)
  • 1% (v/w) osmium tetroxide (OsO 4; Sigma‐Aldrich) in PBS
  • Propylene oxide resin (TAAB Laboratories Equipment)
  • Incubator (set to 60°C)
  • Microtome (Reichert Ultracut E)
  • Carbon‐coated 300‐μm mesh copper grids (Agar Scientific)
  • TEM instrument with imaging modality
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Figures

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Literature Cited

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