1. Overview of the Toxicity Issues with Nanoparticles

2. Designing a realistic test
Meaningful results on the toxicity of nanomaterials are achieved when the conditions of possible exposure are reproduced accurately
How these agent would enter the body?
ACCIDENTALLY
Environmental contamination
Work place exposure
DELIBERATELY INTRODUCED
Medicine applications
Different methods of exposure of the nanoparticle might produce different results
Nature nanotechnology (2009) , Vol. 4, pp 395

3. Influence of Properties on Lung Deposition as well as Toxicity
Ultra-fine or nanoparticles may deposit as aggregates due to high Van Der Waals forces, rather than discrete particles.
If an inhaled particle with a diameter of nm forms an aggregate of 5-10 particle types, in terms of deposition it may have the properties of a nm particle.
Inhaled agglomerates may dissociate when in contact with lung surfactants.

4. Translocation of Probes in the Blood Circulation to Bone Marrow in Rodent Models
PEG-QDots
Metallo-C60 HAS-coated PLA NP PS beads
10 nm nm <200 nm >200 nm
Fast appearance in liver, spleen, lymph nodes and bone marrow (mouse)
Highest accumulation bone marrow after liver, continued increase in bone marrow and decrease in liver,
Significant accumulation in bone marrow after liver
Rapid passage through endothelium in bone marrow, uptake by phagocytizing cells in tissue (mouse)

5. Lung toxicity

6. Cardiotoxicity

7. Nanoparticle Deposition

8. (a) SEM image of lung trachea epithelium, showing cilia (mucociliary escalator), (b) Human alveolar macrophage (center, yellow) phagocytosis of Escherichia coli
(multiple ovoids, green), together with a red blood cell (red). (c), (d) Alveoli in the lung.
(e) Deposition of inhaled particles in the human respiratory tract versus the particle diameter, after.

9. Instillation of CNT’s in Rats
Rats that were instilled with high doses of SWCNT’s died of respiratory blockage rather than pulmonary intoxication
Micrograph of Lung Tissue in Rats
Methods
Four kind of particles including SWCNT’s
Pressurized Intratraqueal instillation
Tracking of alveolar response
Observation periods at 24h, 1 week, 1 month and 3 months
Results
The picture shows that the respiratory airways are mechanically blocked by carbon nanotubes. This led to the asphyxiation of 15% of the test population
Inflammation, no cytotoxicity
Toxicological Sciences (2004) , Vol. 77, pp 117

10. Inhalation of CNT’s in Rats
Exposing rats to air contaminated with CNT’s led to immune-suppression
Mechanism for Immune-suppression by CNT’s
Methods
Air contaminated with low concentration CNT’s
Exposure 6h per day during 14 days
Tracking of proteins and immune response
Results
A signal, likely TGFβ, is released when the carbon nanotube is inhaled. This was tested by isolating the BALF protein from both exposed and control rats. It was shown that the protein from exposed mice cause immune-suppression
Immune-suppression
Nature nanotechnology (2009) , Vol. 4, pp 451

11. Fullerene Disruption of Cell Membranes
Unbiased MD simulations show that the fullerenes easily pass through the lipid head group, to further diffuse slowly within the bi-layer region
Migration of a Single Fullerene
Average penetration time is 500 ps
Pore formation appears not to be induced by the presence of fullerene
Migration of a Fullerene Aggregate
Average penetration time is 1 µs
Nature nanotechnology (2008) , Vol. 3, pp 363

12. Fullerene Disruption of Cell Membranes
The presence of fullerenes inside the membrane appears to barely affect the structure of lipid bilayer
Snapshot of Fullerene Positions Inside the Membrane
Change in the Order Parameter at Different Positions of the Fullerenes
Outside of Membrane
Fullerene and fullerene aggregate are kinetically and thermodynamically favored to locate near the center of the membrane
The mechanism of cell disruption due to mechanistic damage of the cell membrane by the fullerene is discarded
Possible mechanism of disruption of cell function is through the change of elastic properties of the membrane
Nature nanotechnology (2008) , Vol. 3, pp 363

13. Silver Nanoparticles Toxicity
The toxicity of silver nanoparticles was tested using embryos of zebra fish
TEM images of Ag Nanoparticles
starch
BSA
Optical characterization
Two kind of nanoparticles were used. One capped with BSA and the other one with starch
Extent of toxicity is to be measure in term of mortality rate, hatching, heart rate and abnormal phenotypes
The coating of the nanoparticle confer them the desired solubility and stability properties in water
Nanotechnology (2008) 4, 873

14. Silver Nanoparticles Toxicity
The toxicity of silver nanoparticles was tested using embryos of zebra fish
Normal Embryo
Malformed Embryo
Dead Embryo
The zebra fish eggs were taken to a 96-well plate, and a solution of silver nanoparticles at different concentrations was added to each well.
The images show the appearance of normal, malformed and dead embryos. Visual counting was made
Nanotechnology (2008) 4, 873

15. Silver Nanoparticles Toxicity
It was found that the silver nanoparticles were able to trespass the embryo barrier and settle inside, thus causing the effects to be observed
TEM Mitochondria
TEM Nucleus
EDS of embryo
Nuclear deposition is believed to create a cascade of toxic events leading to DNA damage and similar ones
It is possible that the nanoparticles may enter the cells through many routes. Among them, endocytosis through the embryo wall is more likely
Nanotechnology (2008) 4, 873

16. DNA Damage of Cobalt-Chromium NP
The increasing use of nanoparticles (NP) in medicine has raised concerns over their ability to reach privileged sites in the body.
CoCr NP can be created by wear of orthopedic joint replacements
Schematic of exposure setup
The indirect effect of CoCr nanoparticles on human fibroblasts cells was evaluated. The fibroblast cells were protected by a cell barrier made out of BeWo (a human choriocarcinoma). The set up models the protein transport through placenta and similar barriers
Nature nanotechnology (2009) 4, 873

17. DNA Damage of Cobalt-Chromium NP
Metal was observed to be internalized in the barrier, but curiously there were not morphological signs of cell death in the barrier
TEM image
XEDS
Accumulation of nanoparticles is revealed by TEM images
XEDS shows cobalt concentration inside the barrier decreases
No cellular death
Ions are found to trespass the barrier, and small concentrations of metal are also found past the barrier. However, the damage to the cells underneath is larger when the barrier is present. Therefore, a mechanism involving the barrier must exist to cause the DNA damage
Nature nanotechnology (2009) 4, 873

18. DNA Damage of Cobalt-Chromium NP
The DNA damage of the cells below the barrier occurs through a chain of events starting with the damage of the mitochondria in the top layer of the cell barrier which end up in secretion of ATP from the bottom layer to the fibroblasts
DNA Damage Mechanism Schematics
Nature nanotechnology (2009) 4, 873

19. NSF and Why Gd Should be Avoided
The most commonly pursued MR contrast agents have serious issues!!
Issues
Toxicity: Recent discovery of NSF associated with Gd based MRI agents (1997)
NSF lawsuit commercial
BOXED WARNING:
NEPHROGENIC SYSTEMIC FIBROSIS (NSF)
Gadolinium-based contrast agents increase the risk for nephrogenic systemic fibrosis (NSF) in patients with:
• Acute or chronic severe renal insufficiency (glomerular filtration rate <30mL/min/1.73m2) or
• Acute renal insufficiency of any severity due to the hepato-renal syndrome or in the perioperative liver transplantation period.
Patient with NSF

20. More Kinetic and Thermodynamic stability Open chain -MEDIUM RISK
Gadolinium characteristics
Macrocyclic-LOW RISK
More Kinetic and Thermodynamic stability
Rare-earth lanthanide metal (atomic #64)
Gd3+ highly toxic, so bound to chelate
Chelate prevents dissociation in vivo
Eliminated unchanged in glomerular filterate
Patients with impaired kidney or renal function is affected
*Most reported cases of NSF are with a nonionic, linear chelate
Stability: Linear nonionic complexes <linear ionic <cyclic ones.
HIGH RISK-Open Chain
IONIC
NON-IONIC
Open chain -MEDIUM RISK
IONIC

21. Understanding the Properties Before Using In Vivo
Physical
Characterization:
– Size
– Size distribution
– Molecular weight
– Morphology
– Surface area
– Porosity
– Solubility
– Surface charge density
– Purity
– Sterility
– Surface chemistry
– Stability
– No of ligands, CAs, drugs
NCL assay cascade
In Vitro:
– Binding
– Pharmacology
– Blood contact properties
– Cellular uptake
– Cytotoxicity
In Vivo:
– Absorption
– Pharmacokinetics
– Serum half-life
– Tissue distribution
– Excretion
– Safety
Plasma PK profile/
Tissue distribution
(Liver, lungs, kidney, heart, spleen)

22. Another Aspect to Worry Complement Activation
NP decorated with Gd generates complement activation by eliciting immune response
Complement Activation
100,000 Gd
Cell Lysis
Failed Phase I Clinical Trial (Kereos, Inc.) X
>100K Gd on the surface
Needed for detecting angiogenesis
Source: wiki
Solution: Increase safety by replacing or reducing Gd

23. USA Agencies Efforts NSF EPA DoD NTP DoE NIH NIST
Toxic effects of nanoparticles: nanoparticles in air pollution, water purification, nanoscale processes in the environment
EPA
Toxicology of manufactured nanomaterials: fate, transport and transformation, human exposure and bioavailability
DoD
Toxicological properties of nanomaterials: computational models that will predict toxic, salutary and biocompatible effects based on nanostructured features
NTP
Potential toxicity of nanomaterials: titanium dioxide, several types of quantum dots, and fullerenes
DoE
Transport and transformation of nanoparticles in the environment: exposure and risk analysis, health effects
NIH
Nanomaterials in the body: cell cultures and laboratory use for diagnostic and research tools
NIST
Developing measurement tools: tests and analytical methods
EPA and Nanotechnology: strategy, responsibilities and activities, April 2006