Nanotechnology Risks

1. Nanotechnology Risks BIOE298DP

2. How Do we Get Nanoparticle Exposure?

3. Human and Ecological Exposure HUMAN EXPOSURE HUMAN EXPOSURE ECOLOGICAL EXPOSURE

4. Particle Size by Definition • Nano = Ultrafine = < 100 nm (Conventional) • Nano = <10 nm (unique quantum and surface-specific functions) • Fine = 100 nm - 3 m • Respirable (rat) = < 3 m (max = 5 m) • Respirable (human) = < 5 m (max = 10 m) • Inhalable (human) = ~ 10 - 50 m

5. Particle Scale PM 10 Ultrafine Respirable PM 2.5 Nanoparticles 10 mm 1 mm 1 nm 10 nm 100 nm

6. “With Great Power Comes Great Responsibility” The same properties making nanomaterials so interesting can make them potentially harmful Gold Nanoparticle Catalyst • Enhanced reactivity • Increased surface to mass ratio • Enhanced permeation • Relevant quantum effects www.sciencedaily.com • Previously unknown forms of common materials Previously known as a fairly inert material, gold is highly active in its nanoparticle form

7. Schematics of Human Body with Pathways of Exposure, Affected Organs and Associated Diseases from Epidemiological, in vitro and in vivo studies

8. Exposure Scenarios Literally ALL SUBSTANCESin the world are toxic to plants, animals and humans at some exposure levels LONG-TERM ROUTES SHORT-TERM ROUTES Soil adsorption Inhalation in gas phase Skin contact in solution Water dissolution Oral ingestion Biodegradation issues Nature Biotechnology (2003) , Vol. 21, pp 1165

9. Considerations A number of points arises when determining the method to test for toxicity: Dosage and formulation of test material Duration and route of exposure What to look for References and standard materials for comparisons Biological species to be subject of tests Nature nanotechnology (2009) , Vol. 4, pp 395

10. Understanding the Properties Before Using In Vivo NCL assay cascade 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 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

11. Inter-particle Forces and Surface Chemistry will be Influenced by Size and Whether Particles are Individual or Aggregates & Agglomerates Mechanical interlocking Single particle Capillary (surface tension) Van der Waals (cohesive force α 1/d**2) Chemical bonds Equivalent diameters of 10-1000x are common Equivalent dia. ~2 x Settling velocity ~3-4 x

13. PART I Overview of the Toxicity Problem of Nanoparticles

14. 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.

15. 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 90-250 nm <220 nm 240 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)

16. USA Agencies Efforts NSF 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 Toxicological properties of nanomaterials: computational models that will predict toxic, salutary and biocompatible effects based on nanostructured features DoD 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

17. PART II Methodology for Toxicity Studies of Nanoparticles

18. 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

19. Example I: 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 The picture shows that the respiratory airways are mechanically blocked by carbon nanotubes. This led to the asphyxiation of 15% of the test population Results Inflammation, no cytotoxicity Toxicological Sciences (2004) , Vol. 77, pp 117

20. 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 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

21. Nanoparticle Deposition

23. PART III Toxicology of Carbon Nanoparticles

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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 Protein CNT 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

31. 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

32. 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 Nanotubes Biodegraded Nanotubes Control 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

33. PART IV Toxicology of Metal Nanoparticles

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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 Patient with NSF 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.

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

43. How Should We Look at the Safety? In vivo STABILITY CELL TOXICITY BLOOD SMEAR SERUM STABILITY Excreted particles? WHOLE BODY CLEARANCE 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 CLINICAL PATH Pham et. al. J. Biol. Chem 286, 123-130 (2011)

44. Stability: In vivo Whole body clearance Mn Mn-Gd Example clinical pathology Clin-path: No impaired kidney/renal function Whole body clearance: Only residual left after 2wks

45. 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)