NMs and Safety Experiences

Fernando Pastrana MD, MSc, PhD.

Departamento de Ingeniería Eléctrica y Electrónica

Alba Avila PhD

Profesor Asociado, Departamento de Ingeniería Eléctrica y Electrónica

Bogotá D.C., February 14 and 15, 2017 1

Nanomaterials in the market

11/03/13 4

Beilstein J. Nanotechnol. 2015, 6, 1769–1780.

Outline

• Nanomaterials in products• From the bench to the market

• Toxic effects of NMs

• NMs characterization• Physic-chemical to biological identity

• Labeled free cytotoxicity assessment

• Implementing a safety culture

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• Oxidative Stress• ROS• NOS

• Inflammation• Interleukins• Complement activation• Neutrophils

• Mechanical Toxicity• Cytoskeleton disruption

Cells toxicity mechanism

Elsaesser, C.V. Howard, Toxicology of nanoparticles., Adv. Drug Deliv. Rev. 64 (2012) 129–37.

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Carbon-based nanomaterials

• Mesothelioma

SiO2nanoparticles

• Platelet aggregation,• Infarcts

• Fibrosis• Hepatic steatosis

Dendrimers

Nanomaterial Organ Toxic Effect Reference

Potential Organ toxicity of NMs

Part. Fibre Toxicol. 6 (2009) 2.

Int. J. Nanomedicine. 7 (2012) 631–9

Arch. Toxicol. 88 (2014) 1939–64

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How a nanomedical product can be in the market?

Concept

Pre-clinical

•In-silicon

•In-vitro

•In-vivo

Clinical trials

• Three phases

Approval

1 Year

3 Years

5 – 7 Years

Aprox 10 Y

Bogotá D.C., February 14 and 15, 2017

10Bogotá D.C., February 14 and 15, 2017

Clinical trials with nano

Source: clinicaltrials.gov acceded on Feb 15 2017

Outline

• Nanomaterials in products• From the bench to the market

• Toxic effects of NMs

• NMs characterization• Physic-chemical to biological identity

• Labeled free cytotoxicity assessment

• Implementing a safety culture

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Physic-chemical properties

Size Biodistribution

Translocation

Biological barriers

Specific surface area

Surface area/volume ratio

Shape and aspect ratio

Fiber like

Planar

Amorphous

Spherical

Surface reactivity

Surface chemistry

Photocatalytic activity

ROS production

Composition Oxides

Polymers

Carbon

Crystalline structure

Defects

Aggregation & Agglomeration

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Biological Characterization

Sterility Endotoxin detection

Microbial contamination

Bacterial Contamination

Mycoplasma contamination

Hemocompatibility Hemolytic properties

Platelet aggregation by cell counting

Platelet aggregation by light transmission

Complement activation In-vitro immunology

Leukocyte proliferation

Chemotaxis

Phagocytosis

Cytokines, Chemokines and Interferons

Toxicity Oxidative stress

Necrosis

Apoptosis , viability

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Toxicity

Biological Characterization

Oxidative stress Viability Apoptosis

DHE

CellROX

MTT

Calcein

Anexin V

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Safety assessment or nanomaterials interference

Biological Characterization

Agglomeration effectsInterference with assay components

- Increase reagent biotransformation- Bioactive protein sites sequestration

Assays dependence on optical - NMs blocking the light path- NMs reflecting the light path- NMs fluorescence at the same

wavelength than assay dyes

NR: neutral redMTT: tetrazolium to formazan96AQ: MTS to formazanaB: rezasurin to red resorufinCTB: CellTiter-BlueCTO: CytoTox OneToxicology and Applied Pharmacology 234 (2009) 222–235

SWCNT

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Confusing principle

Bogotá D.C., February 14 and 15, 2017

Outline

• Nanomaterials in products• From the bench to the market

• Toxic effects of NMs

• NMs characterization• Physic-chemical to biological identity

• Mechanical indicators-Labeled free cytotoxicity assessment

• Implementing a safety culture

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New labeled free approaches

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Impedance (electrical current)

high sensitivity from no label

Real time recording

Microfluidics (improve current biochemical label assays)

Improve of ENM dispersion, better correlation dose toxicity, physiological environment

Biological Physiology

Multi (Proteo and metabo) nomics

specific effects, cellular pathways

Identification of the individual pathways involves on cytotoxicity

Atomic force microscopy (mechanical response to toxicity

Corroboration of the observed results by immunofluorescence techniques

No label required, in-vivo and environmental assessment

Nanomaterials 2015, 5(3), 1181-1199

Mechanical cellular response

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Pastrana et, al

Cell viabilityAssay (MTT)

Basic Logic Gate diagram of Cytotoxicity assessment of NIH3T3 Exposed to 0.05wt% CBNs after 24h

No toxicity, cells viableCell viability

(Trypan Blue)

Cell viabilityAssay (Live/dead) No toxicity

orToxic NMsCellular stress

(CellROX®)

Cell viabilityAssay (MTT)

Cell viability(Live/dead)

Cellular stress (CellROX®)

Mechanical Toxicity(AFM)

Cytotoxic nanomaterial

Pastrana et al, thesis 2016

31Bogota, February 14 and 15, 2017

1 terminal

2 terminal

3 terminal

Number of terminals:

SGM SMM

CAFM SSRMPFM

SCMEFM-SKFM

Electrical Measurements

32Bogota, February 14 and 15, 2017

Fundamental concepts on microscopy

DIDACTIC MODELS FOR KIDS

+

+

33Bogota, February 14 and 15, 2017

Clubes de ciencias600 studentes

Ingenieros sin fronteras

Teusaca

Outline

• Nanomaterials in products• From the bench to the market

• Toxic effects of NMs

• NMs characterization• Physic-chemical to biological identity

• Mechanical indicators-Labeled free cytotoxicity assessment

• Implementing a safety culture

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Nano activities distribution at UniandesDistribución de actividades nano en Uniandes

Nano-Risk ChallengeRetos con el Riesgo-Nano¿How to manage the safety of a laboratory where nano-

materials are handled?¿Cómo gestionar la seguridad de laboratorios que sintetizan o manipulan nano-materiales?

¿How to turn research & development processes intoacademic oportunities to construct safety cultutre?

¿Cómo convertir los procesos de investigación y desarrollo en oportunidades académicas para construir cultura de seguridad?

¿How to include the emerging risks related to the handlingof nano-materials or nano-composites in the HSE

management?¿Cómo incluir los riesgos emergentes asociados al uso de nano-materiales o nano-

compuestos en la gestión HSE?

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Ti NPZn NPAg NP

Carbon Based Nanomaterials

Potential exposed groups

Potential nano

materials hazards

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ISI and Scopus publication trackSeguimiento de publicaciones en ISI y Scopus

Purchasing department requestSolicitud al departamento de compras

Pre-training surveyEncuesta pre-entrenamiento

• Physics• Chemist• Engineering Dep.

Scenaries and context definitionDefinición de contexto y escenarios

• Engineering barriers• PPE Barriers

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Frequently used nanomaterialNanomateriales mas usados

Risks identificationIdentificación de riesgos

High

Low to Medium

Medium

Medium

High

Low to High

Hazard*

Peligrosidad

*Musee N: Oct 4 2010

MWCNT

Ag Nanoparticles

Corrective controlsControles correctivos

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Safe handling of carbon based nanomaterials’ protocols

Public access to the protocols by intranet

Identify the different CNTs

Explain the potential health risk

Assess the risk according with the CNT & Activities

• Administrative

• Engineering

• Personal protective equipment

• Waste disposal

• Accident management

Suggest the appropriate exposure barriers

web

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Self-assessment

Nanomaterial hazard identification

Exposure probability

• Protective equipment

• Engineered barriers

Safety culture

nanoseguridad.uniandes.edu.co

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NanoRisk App geography

Source google analytics

0

50

100

150

200

250

300

350

400

450

500

ago-15 sep-15 oct-15 nov-15 dic-15 ene-16 feb-16 mar-16 abr-16 may-16 jun-16 jul-16 ago-16 sep-16 oct-16

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Some Strategic Programmes

Thanks

46hf-pastrana122@uniandes.edu.co

Acknowledgements

F. Muñoz . Profesor Ingeniería Química. Universidad de los AndesAlexis Vignes, INERISCandace Tsai, Colorado State UniversityA. Raman, Purdue UniversityAlexander Cartagena, NIH

Fluorescence image of NIH3T3 cell exposed to graphene

3D figure of AFM measurements on NIH3T3 cell

Force map of NIH3T3 cell by AFM

Topographic map of NIH3T3 cell by AFM

ROS RO

S

Protein media adhesion

ROS

F-actin

depolimerization

ROS production

ROS

Surface chemistry• X-ray photoelectron spectroscopy (XPS)

http://www.nuance.northwestern.edu

Sensitivity 0,1%Chemical state infoThickness up to 8nm

Photoelectric effect

KE = hν - (EB+ϕ)

Photoemission

h𝑣

Detector

Probe https://www.helmholtz-berlin.de http://biointerface.org/surface/xps/

Composition• Atomic absorbance spectrometry (AAS)

Lab-training.com

Skidmore's Analytical Interdisciplinary Laboratory (SAIL)

Pastrana et, al

Composition

• Mass spectroscopy

Pastrana et, al

0,00E+00

2,00E+00

4,00E+00

6,00E+00

8,00E+00

1,00E+01

1,20E+01

1,40E+01

1,60E+01

MWCNT [A]

0,00E+00

5,00E-07

1,00E-06

1,50E-06

2,00E-06

2,50E-06

Graphene [A]

Series2DR. DEEPAK BHANOT

Main objectivesObjetivos principales

Provide safe environment to develop nanotechnology activities at Universidad de Los Andes

Protect human health

Protect environment

Protect facilities

Protect institution goodwill

Promote sustainable research and innovation development

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Proteger la salud humana

Proteger en medio ambiente

Proteger la infraestructura

Proteger la reputación de la institución

Promover el desarrollo sostenible de investigación e innovación

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