RNDr. Jaroslav Turánek, CSc.
Toxicology of Nanoparticles -
Nanotoxicology
Veterinary Res. Inst., Brno
„V neustálém cyklu proměn malé
se stává velkým a velké
nepatrným“
Lao‘c
Formation Aggregation Growth Birth
Termination Dissociation Decay Death
“Nothing is more damaging to a new truth than an old error.”
Johann Wolfgang von Goethe
Log length scale showing size of nanomaterials compared
to biological components and definition of “nano” and “micro” sizes
.
10 nm
Comparison of rat macrophage cells size to
nanoparticles size
Microparticles versus nanoparticles
The ISO definition of nanoobjects. Included as nanoobjects are nanoparticles (nanoscale in all
three dimensions), nanofibers (nanoscale in two dimensions), and nanoplates or nanolayers
(nanoscale only in one dimension). * Nanoscale: a size of between 1 and 100 nm.
Nanoobjects
* Chemical composition, purity, impurities
* Particle size and size distribution
* Specific surface
* Morphology (crystalline/amorphous, shape)
* Surface chemistry, coating, functionalization
* Degree of agglomeration/aggregation and particle
size distribution under experimental conditions (for
example, media with/without proteins)
* Water solubility (differentiation between soluble,
metastable, and biopersistent nanomaterials)
* Surface reactivity and/or surface load (zeta potential).
Main characteristics of Nanoparticles
NANOPARTICLE CLASSIFICATION
1. Dimensionality
2. Morphology
3. Structure and composition
Uniformity and agglomeration state
Complexity array of issues sururrounding toxicity of
nanoparticles
Size: 20–50nm enters CNS
<70 nm, able to escape defense system in vivo
High surface to mass ratio
High sharpness, surface charge, conductivity, solubility, durability
and reactivity
Catalytic promotion of reactions
Ability to adsorb and carry other compounds
Ability to escape defense system in vivo
Ability to cross cellular and sub-cellular membranes
Surface coating (e.g., lecithin, albumin)
Enhance uptake by Type I/II pneumocytes
Transcytosis across capillary
Charged particle (higher inhaled deposition)
Unique features of nanomaterials
New in our present time is the variety of additional materials and compounds as well as the
wide range of possible applications, which are expected to soon increase the loads on man,
plants, animals, and environmental compartments and to raise issues of a risk R arising from
exposure E to the new materials and of the biological effect or hazards H that may cause
biological effects. The probability P of processes must also be considered, because a risk
only occurs when there is a certain probability of the development of biological effects.
Assesment of the risk of nanoparticle´s adverese effects
R = Fp{E,H}
SOURCES OF NANOPARTICLES AND THEIR HEALTH EFFECTS
Natural sources of nanoparticles (90%)
Nanoparticles are abundant in nature, as they are produced in many natural processes,
including photochemical reactions, volcanic eruptions, forest fires, and simple erosion,
and by plants and animals, e.g., shedding of skin and hair.
Human activities (10%) —cars, industry and charcoal burning
The most significant components of total global atmospheric aerosols are:
Mineral aerosols primarily from soil deflation wind erosion with a minor component 1%
from volcanoes 16.8 Mt
Sea salt 3.6 Mt
Natural and anthropogenic sulfates 3.3 Mt
Products of biomass burning excluding soot 1.8 Mt, and of industrial sources including
soot 1.4 Mt
Natural and anthropogenic non-methane hydrocarbons 1.3 Mt
Natural and anthropogenic nitrates 0.6 Mt,
Biological debris 0.5 Mt
Extraterrestrial dust
Ten major sources of dust in the world
Kdo umí kráčet, nezanechává stop
Lao´c
Sand storms visualized at macro and microscale
Satellite image showing dust blowing off mainland China over the Sea of Japan and Pacific Ocean
Forest fires and grass fires
Volcanoes
The quantity of particles released into the atmosphere is enormous; a single volcanic eruption can eject up
to 30x106 tons of ash. Volcanic ash that reaches the upper troposphere and the stratosphere the two
lowest layers of the atmosphere can spread worldwide and affect all areas of the Earth for years.
Ocean and water evaporation
Sea spray from ocean waves
Nano-Organisms
Many organisms are smaller than a few microns including viruses 10–400 nm and some
bacteria 30 nm–700 m.
Nanoorganisms or their components - bacteria, viruses, cells, and their organelles.
Cells, bacteria, and viruses are self-organizing, selfreplicating, dissipative structures with a
shorter-lived structure than inorganic solids. Nanoorganisms generally dissipate when their
supply of energy is exhausted. In contrast, nanoparticles are typically inorganic solids that
require no supply of energy to remain in a stable form.
Nanorobots
Organisms in the nanoscale range or producing solidstate
nanoscale debris
Anthropogenic nanomaterials
Humans have created nanomaterials for millennia, as they are by-products of
simple combustion with sizes down to several nanometers and food cooking, and
more recently, chemical manufacturing, welding, ore refining and smelting,
combustion in vehicle and airplane engines, combustion of treated pulverized
sewage sludge, and combustion of coal and fuel oil for power generation.
History of nanomaterials application
History of nanomaterials application
Indoor pollution
Humans and their activities generate considerable amountsof particulate matter indoors.
Nanoparticles are generated through common indoor activities, such as cooking, smoking,
cleaning, and combustion (e.g. candles and fireplaces). Examples of indoor nanoparticles
are textile fibers, skin particles, spores, dust mite droppings, chemicals, and smoke from
candles, cooking, and cigarettes. Poorly ventilated stoves using biomass fuels wood, crop
residue, dung, and coal are mainly responsible for the death of an estimated 1.6x106
people annually, from which more than a half are children under the age of 5.
Indoor air can be ten times more polluted than outdoor air!!!
100 000 chemical components and compounds
Tipical engine exhaust particles consisting of
carbon aggregates around a larger mineral particle
Nanotoxikology
2005-2012 502 301
publications in WOS
2005-2012 14 949
publications in WOS
Statistics on scientific articles published on a) nanomaterials and
b) their toxicity
Nanotoxicology
Nanoparticles arise from a wide variety of natural and
man‐made sources and have a diverse array of biological,
chemical, and physical properties.
The toxicity of these particles can be roughly divided into
two categories:
1) the enhanced delivery of toxic agents
2) toxicity induced the properties of the particle itself
(e.g. size, shape, surface sharpness, solubility)
Toxicity of nanoparticles
In vitro tests
Nanoparticle surface—increased reactivity
Research has shown that it is not the nanomaterial itself but the “corona” that mainly defines
the properties of the “particle-plus-corona” compound. This makes it necessary to understand
not only the nanomaterial but also the nanoparticle environment when testing for
nanotoxicity
Formation of nanoparticle corona: a) “bare” particle, b) nanoparticle in
contact with proteins, c) corona formation. The corona can consist of a “hard
corona”, proteins firmly attached to the surface, or a “soft corona” of proteins
which are only weakly bound to the nanoparticles and form an equilibrium
layer with the surrounding matrix.
In vitro testing
Transport Rates for TiO2.
ISDD calculated fraction of nano and micron scale TiO2 particles delivered to cells over the duration of a
24 hour in vitro study with a media height 3.1 mm. Different rates of particle transport result in different
time-courses for delivery to cells, which is only complete for large particles by 24 hours.
Artificial nanoparticles
• Engineered nanoparticles represent a novel toxicological challenge. They
are completely novel in evolutionary terms, the evidence shows that they
gain can access to the body, particularly through inhalation, and then
translocate within the body to distant sites at low doses.
• A critical step in nanotoxicology is to characterise the nanomaterial under
examination and this is much more difficult than is the case in classical
toxicology because of the multitude of variables in the parameter space.
These include: particle size, roughness, shape, charge, composition and
surface coating. The latter can change depending upon the matrix into
which it is introduced.
• Estimation of nanoparticle “dose” is complex, requiring a number of
direct and/or indirect technologies to determine how many particles are
reaching defined targets. Testing nanoparticle toxicity based on estimates
of realistic human exposure is required instead of testing unrealistically
high nanoparticle doses with no relevance to real-world exposures.
Dose and in vitro toxicology
• Dose In Vitro is Misunderstood, Unmeasured or
Misrepresented
• Particles are not chemicals! They settle and diffuse at very
different rates depending on the density, size and
agglomeration status
• Nominal media doses, the standard, (μg/ml, μg/plate surface)
misrepresent dose by 1 to 6 orders of magnitude
• Nominal media doses cannot be related to target tissue (e.g.
lung, liver, gut) doses from in vitro studies.
Membranes—interface between nanoparticles and cells
• In a cellular context, membranes, which are phospho-lipid bilayers, partition different
intracellular compartments which each have specific functions.
Potential interactions between NPs
and the dividing cel
3D reconstruction showing the interaction of the centrosome,
the DNA, and the microtubules with SWCNTs
The major potential pathways for inhaled NP transport
into the brain
NPs can be carried to the brain via the vasculature, neuronal transport from the olfactory neurons in the olfactory epithelium
of the nose and then through the cribiform plate to the olfactory bulb, neuronal transport from sensory nerves of the nasal
passageways to the trigeminal nerve, or neuronal transport from sensory nerves of the trachea and intrapulmonary airway to
the vagus nerve
Red rhodamine beads in a neuron cell body of the jugular
ganglion demonstrate neuronal transport of particles from the trachea
of a rat after intratracheal instillation of fluorescent beads
Toxic Warnings
1997 - Titanium dioxide/zinc oxide nanoparticles from sunscreen are found to cause free
radicals in skin cells, damaging DNA. (Oxford University and Montreal University) Dunford,
Salinaro et al.
March 2002 – … engineered nanoparticles accumulate in the organs of lab animals and
are taken up by cells…“ Dr. Mark Wiesner
March 2003 - ... studies on effects of nanotubes on the lungs of rats produced
more toxic response than quartz dust.“ „Scientists from DuPont Haskell
laboratory present varying but still worrying findings on nanotube toxicity.
Nanotubes can be highly toxic." - Dr. Robert Hunter (NASA researcher)
March 2003 - Dr. Howard: the smaller the particle, the higher its likely toxicity
and that nanoparticles have various routes into the body and across
membranes such as the blood brain barrier. ETC Group
July 2003 - Nature reports on work by CBEN scientist Mason Tomson that shows
buckyballs can travel unhindered through the soil. "Unpublished studies by the team show
that the nanoparticles could easily be absorbed by earthworms, possibly allowing them to
move up the food-chain and reach humans" - Dr. Vicki Colvin, the Center's director.
Nanoparticles and the environment
• Nanoparticles released into the environment interact with air, water
and soil. This often changes the surface properties of the particles
which can result in particle aggregation or changes in particle
charge and other surface properties. These effects have been
studied in water ecosystems and soil and show the importance of
understanding nanoparticles and their environmental setting as a
“complex” that needs to be looked at in its entirety in order to
understand particle behaviour in the environment. A current debate
addresses whether nanoparticles can cause toxicity as a contaminant
in, for example, soil or water, via a “piggyback” mechanism on
natural organic matter.
Nanoparticles and cells—in-vitro
nanotoxicology
• Unicellular organisms, which ruled the Earth for
approximately the first 3 billion years of life, exist by engulfing
matter, usually particulate, from their immediate
environment.
• There are the two basic mechanisms, phagocytosis and
endocytosis. The former is generally for large particles and
requires a “recognition” step while the latter is for the transmembrane
transport of liquids and molecules.
• Only by understanding particle uptake dynamics and
pathways in a quantitative way, can nanotoxicological
conclusions be drawn.
Mechanisms on the nano-bio interface
• Mechanisms on the nano-bio interface can be either chemical
or physical.
• Chemical mechanisms include the production of reactive
oxygen species (ROS), dissolution and release of toxic ions,
disturbance of the electron/ion cell membrane transport
activity, oxidative damage through catalysis, lipid peroxidation
or surfactant properties. ROS is considered as being the main
underlying chemical process in nanotoxicology, leading to
secondary processes that can ultimately cause cell damage
and even cell death.
• Moreover, ROS is one of the main factors involved in
inflammatory processes
Physical mechanisms at the nano-bio
interface
• Physical mechanisms at the nano-bio interface are mainly a result
of particle size and surface properties. This includes disruption of:
membranes, membrane activity, transport processes, protein
conformation/folding and protein aggregation /fibrillation.
Possible interactions of ENMs with the cell and
subcellular structures.
Schematic diagram of the mechanisms of “primary” and
“secondary” genotoxicity in nanoparticle exposed cells
It‘s only a matter of support: new directions for
advanced intestinal cell models
Microengineered organs-on-chips
Effect of size
Hollow fibre model
The human-on-a-chip concept
Nanoparticles pahtways within the body
Schematics of human body with pathways of exposure to nanoparticles
The Lung: The Main Portal of Entry for Nanoobjects
Inhaled particles that are smaller than 2.5 mm (PM2.5) have access to the alveolar structures
of the deep lung and may, in high doses, induce inflammation. A very small portion of the
nanoparticles can cross the air–blood barrier and will be distributed via the bloodstream
(red). Within the alveoli, most of the particles will be phagocytized by macrophages (purple)
or dendritic cells (yellow) or may also be taken up by epithelial cells (blue).
There are 300 million alveoli to facilitate this gas exchange via diffusion, encompassing a surface area of
approximately 140 m2.
Effect of the particle size on deposition in respiratory tract
Deposition of NP in respiratory tract
Three-dimensional reconstructed CT images of rat thoracic cavities (A) directly and (B) 2
hours after insufflation of nanocluster dry powder. Note that directly after insufflation,
visualization of the respiratory bronchioles and some alveolar structures in the lung
periphery is evident (A). After 2 hours the nanoclusters are cleared from the central
airways, with contrast more localized to the trachea or lung periphery. Nanoclusters
(green); soft tissue (pink).
Toxic effect of asbestos
Carbon nanotubes
Scanning electron micrographs of particulates in tissue
section. (A) Backscatter SEM image of lung section 10 days after
silica inhalation. An alveolar macrophage loaded with fine-sized
silica particles is identified by the arrow.
Uptake of Nanoparticles via the Olfactory Nerve:
Bypassing the Blood–Brain Barrier
Another quite significant uptake pathway is available to nanoobjects
owing to their small sizes. The particles can be incorporated via the
nerve fibers in the area of the olfactory epithelium.
Instillation/inhalation tests on rodents using different particles have
demonstrated that nanoscale carbon particles, gold particles,
manganese oxide particles, and others are conveyed by transsynaptic
transport.
Nanoparticles can reach the brain directly by passing the olfactory
epithelium and the nervus olfactorius located in the roof of the nose.
It is also conceivable that systemic uptakes take place via the nervus
trigeminus und the sensoric nerve fibers in the tracheobronchial tract.
The quantities reaching the brain via the olfactory nerve are very
small; however, they bypass the blood–brain barrier.
Blood-Brain Barrier
Transport through Blood-Brain Barrier
Tight junction
Anatomy of Olfactory bulb
Healthy Skin: An Effective Barrier Against Many Nanomaterials
The corneal layer of stressed or diseased skin is not intact
Stratum corneum
Induction of autoimmune diseases and
hypersensitivity
Penetration of QDots through skin
Confocal scanning microscopy of porcine skin treated with QD 565 for 8 h. (g-i) QDCOOH.
Confocal-DIC overlay with the green QD fluorescence channel shows QD
localization within the epidermis or dermis (arrows). Scale bars equal 50 µm.
Very small particles (<10 nm) are capable of penetrating through to the epidermis or
dermis. Particle surface coatings or functionalization, which are often used to prevent
agglomeration, may strongly influence the penetration.
Volcanoes and health effects
Short-term effects of ash on health include respiratory effects nose and throat irritation,
and bronchitic symptoms and eye and skin irritation
Long-term exposure to volcanic particulate pollution - the barefoot agricultural
populations living in parts of the world containing volcanic soils, such as Africa,
Mediterranean, and Central America. A large percentage of this population is affected by
diseases of lympho-endothelial origin (Podocoinosis, Kaposi´s sarkoma) (crosscomplication
with AIDS)
Liver and kidney fenestrations
Application of nanoparticles in medicine
Normal vs tumour blood vessels
Nanodiamonds
Images show animals injected with 18F-NDs dorsal (A) to ventral (B), with filtered 18F-NDs
dorsal (C) to ventral (D), with 18F-NDs + Tween 80 dorsal (E) to ventral (F), and finally with 18FNDs
+ PEG8000 dorsal (G) to ventral (H). Organs that exhibited an elevated uptake of the
radiolabeled compound are labeled with numbers for easy identification.
Coronal sections of PET images acquired 120 min after
injection of four different ND preparations
L I P O S O M E S
CLASSIFICATION
VIEWED BY CRYOELECTRON MICROSCOPY
Stealth liposomes
Doxorubicin
Penetration of Stealth liposomes into
normal and tumour tissues
Application of in vivo
fluorescence microscopy
Tumour tissue
Normal tissue
Tumour tissue
400nm
80nm
80nm
80nm
Classes of liposomes based on
their functionality
• Conventional liposomes
nonspecific interaction with
melieu
• Stealth liposomes sterically
shielded , low non-specific
interactions, long circulating
• Targeted liposomes specific
interaction via coupled ligand
• Polymorphic-Cationic change
their phase upon inter-action with
specific agents, pH or temperature
sensitive
Liposomal antimicrobial drugs
Amphotericin B
Application of carbon nanotubes
Quatnum Dots
Nanofibres and nano textiles
50 nm
C
D
E F G
B
Functionalised liposomes and
nanofibre textile
Visualizace liposom-DNA depotu fluorescenčním
celotělním zobrazením
Day 1
Day 28
Application of nanosilver
Nano-scaled silver may:
(1) release silver ions and generate ROS
2) interact with membrane proteins affecting their correct function
3) accumulate in the cell membrane affecting membrane permeability
4) enter into the cell where it can generate ROS, release silver ions, and
affect DNA. Generated ROS may also affect DNA, cell membrane, and membrane proteins,
and silver ion release will likely affect DNA and membrane proteins
Ecology
• In the development and commercialization of nanoscale particles
also potential hazards for humans and the environment occurring
during application and production have to be taken into account.
• rainbow trout gill cells with internalized tungsten carbide
Accummulation of NP in aquatic organisms
Daphnia magna exposed to (a) 20 nm fluorescent carboxylated
polystyrene beads (2.64 mg/l for 1 h), (b) 14 nm carbon black
(1 mg/l for 48 h) or (c) 25 nm TiO2 particles (1 mg/l for 48 h).
The life is just a dream and the only reality in it is LOVE
František Bilek
Thank you for your attention
Methods for characterisation of nanoparticles
Separation Techniques: GPC, FFF, Flow Cytometry
Light scattering: SLS/MALS (Rg), DLS (Rh)
Microscopic methods: TEM, SEM, AFM, Confocal microscopy
Single particle tracikng analysis: NanoSight
Conductivity: particle counters (iZON)
Separační rozsah
FFF – velikost analytu
/ Centrifugal FFF
Fiel Flow Fractionation
• Příčný tok pro separaci
• Separace založena na velikosti/MW
• Separační rozsah 103 – 1012 Da, 1 nm – 10 µm
• Temperace kanálu
Princip AF4
LogM
Elution Volume
Eluční pořadí obecně
Eluční pořadí ve FFF
30.11.2014 134
Flow FFF:
VE ~ 1/D ~ Rh
30.11.2014 www.postnova.com 135
Distribuce molekulové hmotnosti (AF4-MALS)
Mp
[kg/mol]
Výrobce
Mp
[kg/mol]
AF4
68 77
127 129
246 213
310 313
659 651
1040 907
1260 1321
2750 2691
5000 462310
5
10
6
0,0
0,2
0,4
0,6
0,8
1,0
DifferentialMassFraction
Molar Mass [g/mol]
PS-standardy v THF
FFF Aplikace
Figure 1. A sensitive signal detector monitors current flow across the nanopore.
Figure 2. Signal Trace with enlarged Blockade Event.
The qNano is a complete
Laboratory of Bionanotechnology
• Laboratory of
Bionanotechnology
Dynamic light scattering
Benefits of sizing by DLS
Pohyblivá zaostřovací čočka umožňuje snímání signálů z různé hloubky kyvety a
tak eliminuje u koncentrovaných vzorků vícenásobný rozptyl (viz obr b)
New dynamic light scattering technology for high sensitivity and measurement at high
concentration (NIBS) (Non-Invasive Back-scatter) technology extends the range of sizes and
concentrations of samples that can be measured.
The sizing capability in the Zetasizer Nano S and Nano ZS instruments detect the scattering
information at 173°. This is known as backscatter detection. In addition, the optics are not
in contact with the sample and hence the detection optics are said to be non-invasive.
Zetasizer Nano ZS, Malvern
Principle Behind Dynamic Light Scattering
Particles, emulsions and molecules in suspension undergo Brownian motion. This is the
motion induced by the bombardment by solvent molecules that themselves are moving
due to their thermal energy. If the particles or molecules are illuminated with a laser, the
intensity of the scattered light fluctuates at a rate that is dependent upon the size of the
particles as smaller particles are “kicked” further by the solvent molecules and move
more rapidly. Analysis of these intensity fluctuations yields the velocity of the Brownian
motion and hence the particle size using the Stokes-Einstein relationship.
D
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Rh
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Dynamic Light Scattering
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Autokorelačná funkcia (8 02.10.07)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
0,1 10 1000 100000 10000000 100000000
0
čas (μs)
korelačnýkoeficient
autocorrelation function
Distribution by DLS
Advantages Dynamic Light Scattering technology
Accurate, reliable and repeatable particle size analysis in
one or two minutes
Measurement in the native environment of the material
Mean size only requires knowledge of the viscosity of the
liquid
Simple or no sample preparation, high concentration,
turbid samples can be measured directly
Simple set up and fully automated measurement
Size measurement of sizes < 1nm
Size measurement of molecules with MW < 1000Da
Low volume requirement (as little as 12µL)
What does Dynamic Light Scattering actually measure?
The diameter that is measured in Dynamic Light Scattering is called the hydrodynamic
diameter and refers to how a particle diffuses within a fluid. The diameter obtained by
this technique is that of a sphere that has the same translational diffusion coefficient as
the particle being measured.
The translational diffusion coefficient will depend not only on the size of the particle
“core”, but also on any surface structure, as well as the concentration and type of ions in
the medium. This means that the size can be larger than measured by electron
microscopy, for example, where the particle is removed from its native environment.
Comparative protein Rh values
Various expressinos and aproximation of protein shape
Shape analysis: shape factor p = Rg/Rh
Light scattering data