nanotoxicology diego a gómez-gualdrón final project nanotechnology chen 689-602 texas a&m...

NanotoxicologyDiego A Gómez-Gualdrón

Final ProjectNanotechnology CHEN 689-602

Texas A&M University

OUTLINE

Overview of the Toxicity Problem of Nanoparticles

Methodology for Toxicity Studies on Nanoparticles

Toxicology of Carbon Nanoparticles

Toxicology of Metal Nanoparticles

Conclusions

PART IOverview of the Toxicity

Problem of Nanoparticles

Nanotechnology Products Consumer products using nanoscale materials have an increasingly presence in the market

Properties of Nanoscale Materials

• Increased surface to mass ratio

• Enhanced reactivity

• Enhanced permeation

• Relevant quantum effects

• Previously unknown forms of common materials

The same properties making nanomaterials so interesting can make them potentially harmful

Gold Nanoparticle Catalyst

Previously known as a fairly inert material, gold is highly active in its nanoparticle form

www.sciencedaily.com

The Nanoparticle Spectra Nanoparticles can be made of any material. However, certain nanoparticles are used with higher frequencyMetal nanoparticles

Carbon Fullerene/Nanotubes

www.wikipedia.com

Metal Oxide nanoparticles

www.ifm.liu.se

Quantum Dots

Toxicological Sciences (2004) , Vol. 77, pp 4-5

Exposure Scenarios ALL SUBSTANCES in the world are toxic to plants, animals and humans at some exposure levels

Inhalation in gas phase

Skin contact in solution

Oral ingestion

SHORT-TERM ROUTES LONG-TERM ROUTES

Soil adsorption

Water dissolution

People working in the nanotechnology industry and consumers of nanotechnology-based products would be the first ones being affected

Biodegradation issues

Nature Biotechnology (2003) , Vol. 21, pp 1165

Nanotechnology Issues

• Lack of specific regulations on nanotechnology

• Non-mandatory reports on toxicity of products

The risk evaluation for exposure to nanotechnology products is hindered by the law-protected secrecy of product formulations

• Old criteria and methods becoming obsolete

Exposure Measurement Initiatives

Also:

Researchers from the National Institute for Occupational Safety and Health determine the concentration of nanoparticles in the air while unloading a reactor for producing metal oxide nanoparticle

Nature nanotechnology (2007) , Vol. 3, pp 635

Research Uncertainty

Fate Model Template for Nanoparticles

A high number of uncertainties in the data might make studies as the one below meaningless

•A study made on Swiss waters have determined the risk quotient for Ag and TiO2 nanoparticles, and carbon nanotubes

Case study

•The risk quotient is the ratio of current concentration to a threshold concentration expected to be harmful

•Contrary to TiO2, Ag nanoparticles and carbon nanotubes showed quotients less than one


Fate models calculate the concentration of nanoparticles in different environmental media and the mass flux between them to assess risks

International Initiative Recently, some agencies have taken some actions to establish regulations to nanotechnology

• US National Nanotechnology Initiative (NNI)

• International Standards Organization (ISO)

• British Standards Institute (BSI)


• Environmental Protection Agency (EPA)

Cover of a nanotechnology Safety workshop proceedings

All these agencies have published reports and guidelines related to the handling of nanomaterials and the research approach to nanotoxicology. However, all of them are voluntary to follow

USA Agencies Efforts

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

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

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

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

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

NIST Developing measurement tools: tests and analytical methods

EPA

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

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

Toxicological Sciences (2004) , Vol. 77, pp 4-5

Research Questions An overview to these reports reveal that the following are the critical issues worth of research

exposure assessment of manufactured nanoparticles

environmental and biological fate, transport, persistence,

and transformation of manufactured nanoparticles

ability to extrapolate manufactured nanoparticle

toxicity using existing particle and fiber toxicological

databases

toxicology of manufactured nanoparticles

recyclability and overall sustainability of manufactured

nanomaterials

PART IIMethodology for Toxicity Studies of Nanoparticles

Considerations A number of points arises when determining the method to test for toxicity:

Duration and route of exposure

Dosage and formulation of test

material

References and standard materials for

comparisons

Biological species to be subject of tests

What to measure?


Designing a realistic test


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

How the nanoparticles would enter the body?

ACCIDENTALLY DELIBERATELY INTRODUCED

• Environmental contamination

• Work place exposure

• Medicine applications

Different methods of exposure of the nanoparticle might produce different results

Rats that were instilled with high doses of SWCNT’s died of respiratory blockage rather than pulmonary intoxication

Example I: Instillation of CNT’s in Rats

Micrograph of Lung Tissue in Rats

Toxicological Sciences (2004) , Vol. 77, pp 117

The picture shows that the respiratory airways are mechanically blocked by carbon nanotubes. This led to the asphyxiation of 15% of the test population

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 Inflammation, no cytotoxicity

Example II: 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

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

Methods• Air contaminated with low

concentration CNT’s• Exposure 6h per day during 14 days

• Tracking of proteins and immune response

ResultsImmune-suppression

Test AssessmentsThe methodology used led to different conclusions on the toxicity of CNT’s. Several criteria must be used to select the meaningful results

What are the realistic conditions of CNT exposure in the work place?


QUESTIONS

• What test simulates a more realistic exposure?

• Are these results applicable to all nanotubes?

• Are exposure concentration realistic?

•Do animal subjects respond similar to humans?

Reliable data to answer these questions can make the difference between deeming CNT’s unsafe or safe

Need for Updating MethodsThe methods to determine toxicity have to keep up with nanotechnology development. Immunological evaluation is an example of an area where nanoparticles interfere with conventional assays


Sources of interference for several assays specific to nanoparticle formulation

The more complex structures and physicochemical properties typical of nanoparticles increases the probability of interference , even more so than traditional pharmaceuticals

Consequence: Modified Tests Developed

Schematics of Rabbit Pyrogen Test

Relevant to nanoparticle-based medical treatments is the potential of fever that would induce organ damage

The test is conducted in two phases. If one animal shows T increase larger than 0.5 K, phase 2 follows suit Nature nanotechnology (2009) , Vol. 4, pp 411

Schematics of Colony Forming Unit Granulocyte-Macrophage Test

Nanoparticle medical treatment poses the risk of nanoparticle agglomeration

The test is conducted is conducted both in in vitro and ex vivo conditions, the results are analyzed and give outputs as the images at the right

Consequence: Modified Tests Developed


Switching to In Vitro TestsTraditionally

In vivoIn vitro

Ex vivo

Current Trend

In vitroIn vivo

Ex vivo

advantages

•Cost effective

•Time effective

•No ethical issues

Primary Assay

Primary Assay

A necessity: Increasing the Diversity

Formulation Tests

Methods Subjects

• Type of nanoparticle

• Concentrations

• Solvent, pH,

• Exposure mode

• Exposure time

• Targeted organs• Additives, Coatings

• Rodents• Canines• Swine• Feline• Human Cells

• End points

•In Vitro

•In Vivo

•Ex vivo

Several studies conclude that the response to nanoparticles is too diverse. For instance, dogs and pigs have a higher immune-response to nanoparticles

Improving In Vitro TechniquesSubstituting 2-D in vitro assays for 3-D ones increases the accuracy of the tests. 3-D assays could potentially reproduce better the 3-D structures of cells in in vivo tests

3-D structure of a liver sectionSchematics of a 3-D spheroid scaffold

• Pores (white circles) are created in a hydrogel matrix using a colloidal crystal template. The 5µm spheroid liver cells (grey circles) are grown in the scaffold structure forming a 3-D network of cells

• Concurrent advances in tissue engineering can help to develop more advanced and realistic 3-D assays (introduction of veins, conducts, macrophages, etc…)


advantages•Representation of complex cell-cell and cell-matrix

interactions

3D vs. 2D assays: Quantum Dot ToxicityQuantum dot toxicity in liver cells was lower when measured using the 3D scaffold technique, as determined by the cell death rate after exposure to CdTe nanoparticles

Small 2009, 5, pp 1213-1221

3D scaffold micrograph3D 2D

Dead cells are identified with red color after a staining assay. Despite some cell death in the spheroid surface, the death rate is much higher in the 2D tissue

Results in 3D culture correlate much better to animal in vivo studies

Assay Unification IssuesNo consensus in the toxicity particular nanoparticles calls has been achieved due to the lack of unification of toxicity tests.

Summary of toxicity results in CdSe QD

Environmental Health Perspectives 2006, 114, pp 165-172

Other Alternatives: Ab initio SimulationsThe toxicity of nanoparticles (NP) depends on its physical chemical interactions with gases, liquids and other nanoparticles surrounding them. This can be studied using molecular simulations

Interaction of Water Molecules with NP


Recent Example Studies

• Surface reactivity of ferrihydride NP assembled in

ferritin (and iron storage protein)

• Nanotoxicological implications of oxygen adsorption at silver

surfaces

The specific and quantitative knowledge obtained from theory and simulation can help building predictive models and algorithms for assessing the likelihood of toxicity in various natural environments

Example I: Anatase (TiO2) toxicityTitanium dioxide (TiO2) a powerful photocatalyst coming in three forms; rutile, anatase and brookite. Anatase form has raised concerns on toxicity

Photocatalytic activity of anatase produces the

reactive (toxic) oxygen species

The (001) anatase surface has been shown to be particularly reactive

The number of (001) facets in an anatase NP

depends on NP size and T

Density Functional Theory (DFT) can predict the

number of these facets depending on size and T

Thought ProcessResults

Number of (001) Facets vs. T and Size

ACS Nano (2008) , Vol. 2, pp 2237

The toxicity of the anatase NP is expected to increase with T and decrease with size

PART IIIToxicology of Carbon

Nanoparticles

Fullerene Disruption of Cell MembranesSome 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

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


Proposed Toxic Mechanisms

•Membrane Damage

•Disruption of Membrane Elastic Properties

•Chemical Interaction

Fullerene Disruption of Cell Membranes

Migration of a Single Fullerene

Migration of a Fullerene Aggregate

Unbiased MD simulations show that the fullerenes easily pass through the lipid head group, to further diffuse slowly within the bilayer region


Average penetration time is 500 ps

Average penetration time is 1 µs

Pore formation appears not to be induced by the

presence of fullerene

Fullerene Disruption of Cell MembranesThe 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

CNT Disruption of Pleural TissueDue to similarities with asbestos fibers, carbon nanotubes toxicity is usually related to pulmonary illnesses

Setup for aerolization of MWCNT’sBefore and After Aerolization TEM image

of MWCNT’s


MWCNT’s are aerolized as mice inhale the aerosol to reproduce doses of 0.2 – 4 mg (MWCNT)/kg (mice)

The exposure time was 6h, and the subsequent retrieval of pulmonary tissue was made after 1 day, 2 weeks, 6 weeks and 14 weeks

CNT Disruption of Pleural Tissue

Macrophages (MØ) ‘eating’ the CNT’s TEM image of CNT’s in sub-pleural cells

After just one day of exposure, carbon nanotubes make their way to several lung structures. They are typically engulfed by macrophages


The presence of the carbon nanotubes in the lung tissues is likely to trigger and immune response. Since the nanotubes are trapped by the macrophages the migration of the latter along the lung tissues spread the nanotube to other locations


Different Transversal Sections of Lung Tissue

The immune response to the presence of the nanotubes was evaluated by counting the number of mononuclear cell aggregates (macrophages, lymphocytes, etc)


Aggregate Counts

There is a correlation between the number of aggregate counts and the dosage of carbon nanotube to the mice. Also, it is seen that carbon black (CB) does not trigger as a strong response as CNT’s


Stain Assay Revealing Fibrosis

The immune response to the presence of the nanotubes was evaluated by counting the number of mononuclear cell aggregates (macrophages, lymphocytes, etc)


Fibrosis Detailed Images

After two weeks of exposure fibrosis is detected in the sub pleural tissue, as opposed to the control. The detailed image of the fibrosis lesion reveals the correlation with the macrophages and the presence of the CNT’s

control

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

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 %

Fluorescent Image of Culture PI Stained Dead Cells

Langmuir (2007) , Vol. 23, pp 8671

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 biodegradationNanotube Solutions Time-evolution of IR spectra Time-evolution of Raman 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 Toxicity Molecular simulations are used to get insights on the biodegradation mechanism of hMPO on 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

protein

CNT

Attachment of CNT to the protein

Residual Groups Attacking the Nanotube

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


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

Superoxide MappingQuantitative Analysis of Superoxide

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


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

NanotubesBiodegraded Nanotubes Control

PART IVToxicology of Metal

Nanoparticles

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 NP Despite the presence of the cell barrier the cell underneath were affected by the presence of the metallic nanoparticles as determined by various methods

Electrophoresis of fibroblast cells

Electrophoresis results reveal DNA damage of the fibroblast cells, as revealed by the difference e in the bands of the control hydrogel and the one corresponding to the assay with CoCr NP. Other functions were also affected such as the frequency of mitosis


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

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


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


Silver Nanoparticles Toxicity The toxicity of silver nanoparticles was tested using embryos of zebra fish

Nanotechnology (2008) 4, 873

Two kind of nanoparticles were used. One capped with BSA and

the other one with starch

TEM images of Ag Nanoparticles

starch BSA

The coating of the nanoparticle confer them the desired solubility and stability properties in water

Extent of toxicity is to be measure in term of mortality rate, hatching, heart rate and

abnormal phenotypes

Optical characterization

Silver Nanoparticles Toxicity The toxicity of silver nanoparticles was tested using embryos of zebra fish


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.

Normal Embryo

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

Dead EmbryoMalformed Embryo

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


It is possible that the nanoparticles may enter the cells through many routes. Among them, endocytosis through the embryo wall is more likely

TEM Mitochondria TEM NucleusEDS of embryo

Nuclear deposition is believed to create a cascade of toxic

events leading to DNA damage and similar ones

Silver Nanoparticles Toxicity Toxicity End Points

Toxicity end points reveal a concentration-dependent

occurrence of negative events such as death

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


Model of Au-NP Transfer in Sea Ecosystems It is shown that gold nanorods can easily pass from the water column to the marine food web. The study raises awareness due to the debatable toxicity of gold nanoparticles

TEM Gold Nanorods Tidal Marsh Creek Simulated Mesocosmos

A series of estuarine mesocosmos were modeled using tanks containing sea water, sediment, biofilm, plant species and dosed with gold nanorods

Nature Nanotechnology (2009) 4, pp 441-444

The tides were driven using submersible pumps set to timers

Model of Au-NP Transfer in Sea Ecosystems Results shows the recovery of nanoparticle by the biofilm and sediment. This occurs despite the fact the biofilm accounts for less than 0.5 % of the bio mass of the system

Nano-Gold Distribution

A possible explanation for the affinity of marine biofilms and gold is the latter affinity for the negative charge of monomers constituting the polysaccharides and proteins of their structure

PART VConclusions

Past and Current Work

Almost without exception, every single study of nanoparticle toxicology issues a warning to the exposure to all the various nanoparticles until more conclusive studies can be made

There exists reports establishing guidelines and ground rules for nanotechnology research. These have been issued by governmental agencies worldwide. However, these norms are advisory, not mandatory

There has to be compromise between testing all the possible scenarios for each nanoparticles, and creation of standards to unify tests

Past and Current Work

Joint efforts by in vivo, ex vivo and in vitro tests, and theoretical studies can help putting together all the pieces of the puzzle to evaluate and understand the mechanisms behind harmful effects of nanoparticles

Nanotechnology is a double-edge sword, the same novel properties making nanoparticles attractive, makes them potentially toxic. Particular care must be taken in nanomedicine, since in this area is where greater exposure would be present

Future Work and Challenges

Completing the whole spectrum of toxicity for all the existent (and potentially new) nanoparticles

Modifying existent and create new assays that are up to the challenge of determining nanoparticle-related toxicity

Issuing new laws that compel companies the exertion of ‘responsible’ nanotechnology that facilitate the implementation of accurate fate models

Determine the exact origin of the toxicity of each nanoparticle to make the pertinent modifications in order to obtain safer products and technologies

?QUESTIONS

nanotoxicology diego a gómez-gualdrón final project nanotechnology chen 689-602 texas a&m...

Documents

concentration of nanoparticles

ag nanoparticles

certain nanoparticles

metal oxide nanoparticles

nanotechnology industry

consumers of nanotechnology

university slide

market slide