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    Cytotoxicity and Inflammatory Effect of Silver Nanoparticles
    in Human Cells
     
     
    Cytotoxicity and Inflammatory Effect
    of Silver Nanoparticles
    in Human Cells
    Jeong-shin Park, Na Mi Yu, Jinwoo Cheon and In-Hong Choi
    Department of Microbiology, College of Medicine;
    Department of Chemistry;
    Nanomedical NCRC, Yonsei University, Seoul, Korea
     

    1. Approaches to practical toxicology tests

    to assess nanoparticles

    1. Cytotoxicity and inflammatory effects

    of silver nanoparticles
     
    Nanoparticles and toxicity assay
     
    The rapidly developing field of nanotechnology will result
    in exposure of nanoparticles to humans via several routes
    (e.g., inhalation, ingestion, skin, etc.). Nanoparticles can
    translocate from the route of exposure to other vital
    organs and penetrate cells.

    • Toxicity studies to determine the deleterious effects of

    nanoparticles on living cells are required.

    • Due to the nanosize and the nature of agglomeration,

    simple standard methods to characterize the biological
    effects of nanoparticles are currently unavailable.

    • In this study, practical information regarding the optimal in

    vitro tests for nanotoxicity were evaluated.
     
    Silver nanoparticles
    03/19
    Antimicrobial applications
     
    Ink
     
    Cosmetics 200nm 200nm 500nm
    20 nm (synthetic)
    180 nm
    (commercial,
    Aldrich)
     
     
     
     

    Biological tests  Inflammation ØØ Annexin staining,caspase activation ØØCytokine production,activation of 
    Signaling molecule ØØROS ØØCytotoxicityØØ MTT/CCK-8 ØØ Establishment of in vitro toxicity assay ØØ Identification of mechanisms for toxicity and inflammation
    Synthesis 
    Production & characterization
    of physical and chemical properties

     
     
    In vitro tests for nanoparticles
     

    ISO/TC229 
    • OECD
    • U.S NCL
    Review in vitro
    methods
    • Production of 
    diverse particles
    (size, surface)
    • Assess biological
    activities
    Assess toxicity tests
    Understanding of proper 
    methods for nanoparticles
    Establish proper
    methods

     
    Exposure routes of nanomaterials
       Skin      Respiratory trac   Immune System
     

      Cell line Origin Characteristics  
    Respiratory A549 Lung epithelial Proper for cytotoxicity  
    BEAS-2B Bronchial epithelial 
     
    Proper for cytokine Production
    Immune U937 
     
    Macrophage Proper for cytotoxicity and 
    cytokine production
     
    Skin SK-Mel Skin epithelial Proper for cytotoxicity and 
    cytokine production
     
      A375 Skin epithelial Too fast growing  

    Standard toxicology tests and silver nanoparticles
     

    In Vitro 
    Immunology
    Properties
    (Blood
    contact
    Hemolysis Release of 
    hemoglobin
    Standard Proper 
     
    Complement 
    activation
    Activation of C3 
    complement
    Standard Inappropriate
    Leukocyte 
    proliferation with
    mitogen
    stimulation
    Standard CCK-8
    In Vitro 
    Immunology
    (Cell-based
    assays)
    Leukocyte 
    proliferation
    Zymosan assay Standard Proper
    Cytokine 
    production
    Standard Proper
    Phagocytosis     Proper
    Cytokine induction
    Toxicity Oxidative stress Detection of ROS Standard CCK-8
    Cytotoxicity (necrosis) Cell viability and 
    mitochondrial
    integrity
    Standard Annexin-V
    Cytotoxicity 
    (apoptosis)
    Activation of 
    caspase 3
    Standard  
             
    Targeting Cell 
    binding/internalization
    N/S N/S TEM, confocal 
    microscope or other
    methods

     
    Characteristics specific to metal nanomaterials
     
    Nanoparticles larger than 100 nm tend to aggregate relatively quickly in vitro when compared to nanoparticles smaller than 100 nm. Fresh samples
    within two weeks after synthesis is recommended for tests.

    Each standard toxicology method must be verified before use. (ex. interference with a specific wavelength, electrophoresis)
     
    Flow chart for nanotoxicity tests
     
    Small –Nano Particle Size 100nm-Large
     
    Small–Analysis Of Biological — Nano Particle Size 100nm
     
    – Particle size
    – Cytotoxicityroperties
    – Apoptosis
    – Cytokine production
    – Hemolysis
    – Leukocyte proliferation
    – ROS production
     
    Large— Analysis of chemical/physical
    properties
    – Aggregation
    – Particle size
     
    Cytotoxicity of silver nanoparticles
     
    20 nm
    Cell viability (%)  
    Conc. (μg/mL)
                                                   
     
                                                                                                                                                      Cell viability (%)
    Conc. (μg/mL)
     
     
    SK-Mel28 (skin) A375 (skin) A549 (lung)
     
    Summary
     
    In human cells, epithelial cells from skin or lung, and macrophages, 5 nm and 20 nm silver particles induced stronger cytotoxicity and ROS synthesis than 80 nm
    particles did.

    • 5 nm and 20 nm silver particles induced chemokine production, mainly IL-8, MIF and RANTES, while proinflammatory cytokines, IL-1, IL-6 and TNF-α were not induced significantly in the same conditions.
    • Some MAP kinase signaling pathways were activated during exposure to silver nanoparticles at lower —concentrations which do not induce cytotoxicity

     
    The toxicity and inflammatory effects of nanoparticles are dependent on their size. In silver nanoparticles smaller than 20 nm induce cytotoxicity significantly in vitro.

    • Nanoparticles induce inflammatory immune responses at lower concentrations and chemokines are the major cytokines induced at early stages of exposure to silver