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PART II Overview of the Toxicity Issues with Nanoprobes. 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.

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Overview of the Toxicity Issues with Nanoprobes

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 50-100 nm forms an aggregate of 5-10 particle types, in terms of deposition it may have the properties of a 200-500 nm particle.
  • Inhaled agglomerates may dissociate when in contact with lung surfactants.
Translocation of Probes in the Blood Circulation to Bone Marrow in Rodent Models


Metallo-C60 HAS-coated PLA NP PS beads

10 nm 100 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)

(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.

instillation of cnt s in rats
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


  • 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

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

inhalation of cnt s in rats
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


  • Air contaminated with low concentration CNT’s
  • Exposure 6h per day during 14 days
  • Tracking of proteins and immune response


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


Nature nanotechnology (2009) , Vol. 4, pp 451

carbon nanotubes germ killers
Carbon Nanotubes: Germ Killers

Carbon nanotubes were shown to reduce the viability of E. Coli culture, revealing the germicide effect of pristine carbon nanotubes

SEM image of a normal E. Coli Culture

SEM image of a E. Coli Culture with SWCNT’s

The carbon nanotubes were grown using a cobalt-containing catalyst on a silica support .

The nanotubes were washed and stripped of metal traces and used for the culture.

Staining assays are able to tell the live cells from the dead cells

Langmuir (2007) , Vol. 23, pp 8671

carbon nanotubes germ killers1
Carbon Nanotubes: Germ Killers

Staining assays reveals the high percentage of dead cells due to the presence of carbon nanotubes

PI Stained Dead Cells

Fluorescent Image of Culture

Carbon Nanotube Patch

It is apparent the correlation of the location of the nanotube in the culture and the location of dead cells. Comparison of the fluorescent images for the totality of the cells and the ones dead shows a very high rate of mortality, which was determined to be around 80 %

Langmuir (2007) , Vol. 23, pp 8671

biodegradation of cnt s and toxicity
Biodegradation of CNT’s and Toxicity

Nanotubes are biodegraded using the human myeloperoxidase hMPO, and further used to evaluate their toxicity to evaluate the impact of biodegradation

Nanotube Solutions

Time-evolution of Raman spectra

Time-evolution of IR spectra

The carbon nanotube characteristic bands in the IR and Raman spectra are seen fading as time progresses and their biodegradation advances. The change of these peaks is related to drastic changes in morphology of the nanotubes

Nature nanotechnology (2010) April -Advanced Online Publication

biodegradation of cnt s and toxicity1
Biodegradation of CNT’s and Toxicity

Molecular simulations are used to get insights on the biodegradation mechanism of hMPO on the nanotube.

Attachment of CNT to the protein

Residual groups attacking the Nanotube



A site-localized reaction in which hMPO positive charges favor the binding of nanotubes and radical-supporting aromatic groups participate in the cleavage of the nanotubes

Nature nanotechnology (2010) April -Advanced Online Publication

biodegradation of cnt s and toxicity2
Biodegradation of CNT’s and Toxicity

The likelihood of CNT’s biodegradation in intracellular hMPO is evaluated. CNT’s are injected in cells and the oxidative activity within the cells is tracked

Quantitative Analysis of Superoxide

Superoxide Mapping

The functionalization of carbon nanotubes increased the intracellular superoxide activity, as well as the release of hMPO and peroxide.

Nature nanotechnology (2010) April -Advanced Online Publication

biodegradation of cnt s and toxicity3
Biodegradation of CNT’s and Toxicity

It was demonstrated that biodegraded carbon nanotubes did not cause inflammatory response in pulmonary tissue of mice traqueally instilled with CNT’s


Biodegraded Nanotubes


The images show the formation of granulomas seven days after exposure to pristine carbon nanotubes. On the other hand , the graphs reveal a healthy tissue when the exposure was before biodegraded CNT’s

Nature nanotechnology (2010) April -Advanced Online Publication

fullerene disruption of cell membranes
Fullerene Disruption of Cell Membranes

Some studies has suggested the penetration of fullerenes aggregates through cells, and blood and brain barriers. However, the mechanism is poorly understood

Diffusion Coefficient of Fullerene as It Moves through the Membrane

Proposed Toxic Mechanisms: Disruption of Membrane; Elastic Properties;

Membrane Damage; Chemical Interaction

The results reveal a higher permeability of fullerene through the lipid bilayer is higher than water but lower than hydrocarbon molecules

Nature nanotechnology (2008) , Vol. 3, pp 363

fullerene disruption of cell membranes1
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

Fullerene Disruption of Cell Membranes

The presence of fullerenes inside the membrane appears to barely affect the structure of lipid bilayer

Change in the Order Parameter at Different Positions of the Fullerenes

Snapshot of Fullerene Positions Inside the Membrane

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

silver nanoparticles toxicity
Silver Nanoparticles Toxicity

The toxicity of silver nanoparticles was tested using embryos of zebra fish

TEM images of Ag Nanoparticles



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

silver nanoparticles toxicity1
Silver Nanoparticles Toxicity

The toxicity of silver nanoparticles was tested using embryos of zebra fish

Normal Embryo

Malformed Embryo

Dead Embryo

The images show the appearance of normal, malformed and dead embryos. Visual counting was made

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.

Nanotechnology (2008) 4, 873

silver nanoparticles toxicity2
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

silver nanoparticles toxicity3
Silver Nanoparticles Toxicity

Toxicity end points reveal a concentration-dependent occurrence of negative events such as death

Toxicity End Points

Nanoparticle deposition in the central nervous system could have adverse effects in the control of cardiac rhythm, respiration and body movements

Exposure to silver nanoparticles resulted as well in accumulation of blood causing edema and necrosis

Nanotechnology (2008) 4, 873

dna damage of cobalt chromium np
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

dna damage of cobalt chromium np1
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


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

dna damage of cobalt chromium np2
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

designing a realistic test
Designing a realistic test

Meaningful results on the toxicity of nanomaterials are achieved when the conditions of possible exposure are reproduced accurately

Different methods of exposure of the nanoparticle might produce different results

Nature nanotechnology (2009) , Vol. 4, pp 395

Understanding the Properties Before Using In Vivo

NCL assay cascade



– Size

– Size distribution

– Molecular weight

– Morphology

– Surface area

– Porosity

– Solubility

– Surface charge density

– Purity

– Sterility

– Surface chemistry

– Stability

– No of ligands, CAs, drugs

In Vivo:

– Absorption

– Pharmacokinetics

– Serum half-life

– Tissue distribution

– Excretion

– Safety

Plasma PK profile/

Tissue distribution

(Liver, lungs, kidney, heart, spleen)

In Vitro:

– Binding

– Pharmacology

– Blood contact properties

– Cellular uptake

– Cytotoxicity


A Specific Example for Evaluating Safety

NSF and Why Gd Should be Avoided

The most commonly pursued MR contrast agents have serious issues!!


Toxicity: Recent discovery of NSF associated with Gd based MRI agents (1997)

NSF lawsuit commercial

Patient with 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.

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 glomerularfilterate
  • Patients with impaired kidney or renal function is affected
  • *Most reported cases of NSF are with a nonionic, linear chelate
  • Stability: Linear nonionic complexes

HIGH RISK-Open Chain



Open chain -MEDIUM RISK


Another Aspect to Worry

NP decorated with Gd generates complement activation by eliciting immune response

Complement Activation



  • >100K Gd on the surface
  • Needed for detecting angiogenesis


Cell Lysis

Failed Phase I Clinical Trial (Kereos, Inc.)

Source: wiki

Solution: Increase safety by replacing or reducing Gd

MRI Agents Based on Non-lanthanides

Lanthanide gadolinium is NOT safe: Linked to Nephrogenic Systemic Fibrosis (NSF)

  • Promise
  • Fe(III), Mn(II), Mn(III), Cu(II)
  • Favorable biochemistry
  • Limitation
  • Sensitivity
  • Lack of suitable nano platforms
  • Safety
  • Solution
  • Nano-engineering approach
  • Safe
  • Efficient
  • Commercially amenable

Metals in the form of organometallics

/organically soluble complexes and NP

No metals on the surface as contrary to commonly pursued approaches,


  • Pre-requisites:Bio-metabolizable; 100,000 metal/NP; specificity: nanomolar

Pan et. al. J Am Chem Soc. 2011, 133(24):9168-71.

Pan et. al. J Am Chem Soc. 2008 , 130(29):9186-7.

Pan et. al. ChemCommun (Camb). 2009, 22, 3234-6.

Kim and Pan J Am Chem Soc. 2012, 134(25):10377-80.

SenPan et. al. ACS Nano. 2009, 3(12):3917-26.

Stability: In vivo

Whole body clearance



Example clinical pathology

Clin-path: No impaired kidney/renal function

Whole body clearance: Only residual left after 2wks

Stability: In vitro

Blood smear

Long-term shelf life stability

3 replicate batches of Bi Nanoparticle

Before treatment with NP

  • Remarkable stability when protected from light and kept under inert atmosphere
  • No morphological changes of lymphocytes

After treatment with Bi NP

Pan et. al. AngewChemInt Ed. 9635-9639 (2010)

Complement Activation Assayof MnOL-Gd NC

in vitro hemolysis assay

with human serum

in vivo assay in mouse using


  • A screening assay for the activation of the classical complement pathway
  • Sensitive to the reduction, absence and/or inactivity of any component of the pathway.
  • Functional capability of serum complement components of the classical pathway to lyse human blood.

Collaboratively with Christine Pham, MD

How Should We Look at the Safety?

In vivo





Excreted particles?


In vitro

In vivo

Complement Activation

Understanding the interactions between NPs and the host innate immune response and provide the basis for a systematic structure-activity relationship study

Kidney and renal function


Pham et. al. J. Biol. Chem 286, 123-130 (2011)

usa agencies efforts
USA Agencies Efforts


Toxic effects of nanoparticles: nanoparticles in air pollution, water purification, nanoscale processes in the environment


Toxicology of manufactured nanomaterials: fate, transport and transformation, human exposure and bioavailability

Toxicological properties of nanomaterials: computational models that will predict toxic, salutary and biocompatible effects based on nanostructured features



Potential toxicity of nanomaterials: titanium dioxide, several types of quantum dots, and fullerenes


Transport and transformation of nanoparticles in the environment: exposure and risk analysis, health effects


Nanomaterials in the body: cell cultures and laboratory use for diagnostic and research tools


Developing measurement tools: tests and analytical methods

EPA and Nanotechnology: strategy, responsibilities and activities, April 2006