Biosafety of Nanoparticles · Biosafety of Nanoparticles: Evaluation of Nanomaterials By a Suite of Cellular Assays Mellisa Theodore 1, Nathan Boggs , Bakhtiyor Rasulev PhD2, Jerzy

Biosafety of yNanoparticles:

Evaluation of Nanomaterials By a Suite of Cellular Assays

Mellisa Theodore1, Nathan Boggs1, Bakhtiyor Rasulev PhD2,Jerzy Leszczynski PhD2 and Joany Jackman PhD1

Science & Technology Business Area

Jerzy Leszczynski PhD , and Joany Jackman PhD

1- JHU APL2- Jackson State University

Biosafety of Nanoparticles yOur Approach

Is there a need for concern? Nanoparticles are being used in many industries A variety of shapes and surface chemistry exist at this time- little is known about

the effect of nanoparticles on the host Carbon nanotubes have shown asbestos like health risks Carbon nanotubes have shown asbestos-like health risks Colloidal silver ingestion causes argyria Pharmaceutical, biological research, medical applications

“Wait and see” methodology not acceptable E d f t b th i t t id ti Exposure and fate both very important considerations

What do we want to know? If biomedically-relevant nano-materials can cause damage to cells, cell pathways or

genomic DNA If DNA damage correlates to organ specific effects or cytotoxicity How are we investigating the concerns? Building on a suite of in vitro cellular assays for hazard assessment of nanoparticles

i bi k i i hi h i h l i i bi k f h d

Milton S. Eisenhower Research Center (MERC)

Prescreening biomarkers in vitro which might translate to in vivo biomarkers of hazard assessment

Predicatively modeling cellular effects of nanoparticles Science and Technology Business Area (S&T BA)

2

Need for Concern?Industrial Problem:Industrial Problem: Nanoparticles May Not Be Eliminated by HEPA Filters

High Efficiency Particulate Air Filter99 9% efficient at 0 3 micron

How HEPA filters work:99.9% efficient at 0.3 micron

Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 3

NOTE: Efficiency drops in this size range and …CRITICAL ISSUE

Need for Concern? Nanoparticles: Protection? Fate? Elimination?

Nanoparticle size and respiratory disposition(Maynard and Kuempel, 2005)

…this is size where greatest deposition ...and cannot be eliminated by


g poccurs in lungs filtration in the kidneys

Need for Concern?Medical and Industrial Problem: Nanostructures Have Been Linked ToMedical and Industrial Problem: Nanostructures Have Been Linked To Cancer


Research Question: What is relationship of chemistry, nanoparticle size and shape to risk?

Need for Concern?Hazards of non toxic dust: Effects may not be acute

Pulmonary alveolar proteinosis is a rare disease in which a type of protein builds up in the air sacs (alveoli) of the lungs, making b hi diffi lbreathing difficult

In some cases, the cause of pulmonary alveolar proteinosis is unknown

In other cases it is associated with infection or an immune problem

It also can occur with cancers of the blood system, and afterthe blood system, and after exposure to high levels of dust

This rare disorder generally affects people 30 - 50 years old and is seen in men more often than in


seen in men more often than in women

Science and Technology Business Area (S&T BA) 6

How are we investigating?Suite of Assays and Cells

Assays- Cellular ScreenAssays Cellular Screen Acute measures of toxicity

MTT Assay- cellular metabolic activity Live/Dead Assay- cellular membrane integrity

Long-term measures DNA Ladders; Flow Cytometry (Annexin-V)- apoptosis/ necrosis Flow Cytometry- Cell cycle analysis/ growth kinetics Cytokine Detection Assay- multiplex immunoassay for human cytokines

Cells Lines HL60- promyloblast- peripheral blood THP1- monocytes- peripheral blood HepG2- hepatocyes- liver A549- alveolar epithelial-lung MCF7- breast adenocarcinoma- mammary gland


Nanoparticle Variations Nanoparticles are significantly different in all aspectsNanoparticles are significantly different in all aspects Our studies will consider the following distinctions in particles: Composition Size Size Shape (aspect ratio) Surface charge

Initial Studies: Initial Studies: Gold nanoparticles characterized by NIST 2nm,10nm, 30nm, 60nm

R lt h f H G2 d A549 ll li Results shown for HepG2 and A549 cell lines Follow up Experiments: Comparison of Carbon nanotubes and Fullerenes

I ll i b i


Immune cell responses to incubations ROS Evaluations


Results: MTT ASSAY 24H-72H

Assay results shown up to 72H of incubation with nanoparticles- little death observed compared to positive control (cell death induced by H2O2)

0 7

0.8

0.910nm 100nM

10nm 10nM

( y )

0 35

0.4

0.45

0.5

0.6

0.7

ce a

t 570 10nm 1nM

30nm 100nM

30nm 10nM 0.25

0.3

0.35

ance

0.3

0.4

0.5

bsor

banc

30nm 10nM

30nm 1nM

60nm 100nM

60nm 10nM0.15

0.2

0.25

Abs

orb

0

0.1

0.2A 60nm 10nM

60nm 1nM

Positive Control

N ti C t l 0

0.05

0.1


0 Negative Control 024H 72H

Results: LIVE / DEAD ASSAY 24H-72HAssay results shown up to 72H of incubation with nanoparticles- little death observed compared to positive control (cell death induced by H2O2)

90%

100%

24 H Live Dead‐ 10, 30, 60nm Gold Nanoparticle Incubations

y

90%

100%

72H Live Dead‐ 10, 30, 60nm Gold Nanoparticle Incubations

40%

50%

60%

70%

80%

40%

50%

60%

70%

80%

90%

Fluo

rescen

ce0%

10%

20%

30%

0%

10%

20%

30%

40%

% F

24H 72H


24H 72H

Results:CELL CYCLE EFFECTS


Normal Cell Cycle G2 phase Block G1 Phase Block*

The Inflammatory Response

Fever

Vascular leakage


gIncreased blood flow

Results: 17-Plex Cytokine Assay

Cytokine Concentration Gold Nanoparticle ChallengeCytokine Concentration, Gold Nanoparticle Challenge (72hr)

NC

8 0010.0012.0014.00

pg/m

L)

PC10-10010-1010 1

0 002.004.006.008.00

ntra

tion

(p 10-130-10030-1030-10.00

IL-1

bet

a

IL-2

IL-4

IL-5

IL-6

IL-7

IL-8

IL-1

0

IL-1

2(p

70)

IL-1

3

IL-1

7

G-C

SF

GM

-CS

F

IFN

-g

amm

a

MC

P-1

(MC

AF

)

MIP

-1b

eta

TNF

-al

pha

Con

ce

Cytokine

30 160-10060-1060-1


Cytokines appear to have been induced by Nanoparticles…

Results: 17-Plex Cytokine Assay

Cytokine Concentration, LPS Challange Compared to Gold Nanoparticles

•Comparison to LPS induced

10

15

20

25

entra

tion

(pg/

mL)

NCPC10nm - 100nM10nm- 10nM10nm- 1nM30nm- 100nM30nm- 10nM30nm- 1nM

•Comparison to LPS induced cells shows higher induction in positive controls

0

5

IL-2 IL-12 G-CSF GM-CSF

48H Cytokines

Conc

e

60nm- 100nM60nm- 10nM60nm- 1nMLPS Control

•Cytokine induction in immune cells is much higher than in hepatocytes

Positive Control (10ng/mL LPS from K. pneumoniae)

35404550

g/m

L)

1 hour3 hour6 hour

05

101520253035

Con

cent

ratio

n (p 9 hour

12 hour18 hour


0

IL-2

IL-4

IL-6

IL-8

IL-1

0

GM

-CSF

IFN

-g

TNF-

a

IL-1

b

IL-5

IL-7

IL-1

2(p

70)

IL-1

3

IL-1

7

G-C

SFM

CP-

1(M

CAF

)M

IP-1

b

Results: Prescreening Biomarkers in vitro and Predicative Modeling Investigation of cytokine responses using modeling is necessary

for prescreening of potential biomarkersfor prescreening of potential biomarkers When placed into model Analyzing cytokine assay data- noticed that response values

for 2nm and 30nm particles were less then for 10nm and forfor 2nm and 30nm particles were less then for 10nm and for 60nm

This was noticed for almost all cytokine assays Selected IL-7 assays have the same distribution of values y

(expected that the response value should increase with decreasing or increasing nanoparticle size)

Wanted to find any rule for experimental data distribution and then find parameters that change noticeably with theand then find parameters that change noticeably with the cluster size increasing

By analyzing the data for IL-7 assay -found some interesting distribution of dataM d li Eff t C d t d b


Modeling Effort Conducted by: Interdisciplinary Center for Nanotoxicity ~ Jackson State University

Results: Prescreening Biomarkers in vitro and Predicative Modeling-g gIn silico approaches

SARs: Structure-

Physical Properties Toxicity

SARs: StructureActivity Relationships

QSARs: Quantitative Structure-Activity Relationships E i t l Bi ki tiRelationships Environmental

DistributionBiokinetic

Parameters



Results: Prescreening Biomarkers in vitro and Predicative Modeling

3-dimensional plot of data- difficult to see regularity

M d li Eff t C d t d b



Results: Prescreening Biomarkers in vitro and Predicative Modeling

When plotted on one axis, can see a sinusoidal curve, which is gradually damping

Looked for equation to fit the curve Among several thousands curve fitting functions

we found the following: we ou d e o ow g

Y(IL7)=7.88E-03·(X12·ATAN(X2))+3.64·(COS(X1)

ATAN(X2))+3.64

where Y(IL7) is response variable, IL-7 cytokine assay values, X1 – gold nanoparticle size in nm, X2 – concentration in nM

Need to check robustness of this equation with experimental data for other sizes of gold

inanoparticles

M d li Eff t C d t d b



TEM Images of Gold Nanoparticles10nm 30nm 60nm10nm 30nm 60nm


TEM Images of Gold Nanoparticles: Incubated with cells


Potential uptake of nanoparticles into cells

Conclusions: Well characterized gold nanoparticles have shown little/ no effect in acute

toxicity assays Further testing is scheduled for more cell lines and various types of

nanoparticlesD t f f ll i t ill b il d i t th d t b f Data from follow on experiments will be compiled into the database for continued modeling for prediction of relative risk of well characterized materials

Disruption of cellular pathways and biomarkers of exposure will be used toDisruption of cellular pathways and biomarkers of exposure will be used to develop predictive monitoring tools

Acknowledgements:Acknowledgements: APL: Melissa BetzNIST: Dr. Bryant Nelson, Dr. John ElliotJohns Hopkins University: Microscopy Facility


Johns Hopkins University: Microscopy Facility


References:

Atsuya Takagi, Akihiko Hirose, Tetsuji Nishimura, Nobutaka Fukumori, Akio Ogata, Norio Ohashi, Satoshi Kitajima and Jun Kanno: “Induction of mesothelioma in p53+/− mouse by intraperitoneal application of multi-wall carbon nanotube”: J. Toxicol. Sci., Vol. 33: No. 1, 105-116. (2008)

HEPA Filtration Facts. D. F. Solutions. Minneapolis: 1-2 Maynard, A. and E. Kuempel (2005). "Airborne Nanostructured Particles and Occupational Health." Journal of

Nanoparticle Research 7(6): 587-614Nanoparticle Research 7(6): 587 614 Poland, C. A., R. Duffin, et al. (2008). "Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like

pathogenicity in a pilot study." Nat Nano 3(7): 423-428 Song, Y., X. Li, et al. (2009). "Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma."

European Respiratory Journal 34(3): 559-567 Wick, P., A. Malek, et al. (2009). "Barrier Capacity of Human Placenta for Nanosized Materials." Environ Health Perspect

( )118(3)

Image Sources http://www.atcc.org/Attachments/1761.jpg http://losangelespublicrelations.com/colloidal-silver-blue-man/0781 http://www.pharmacology2000.com/respiratory_anesthesiology/pulmonary_assessment/pulmonary_assessment1.htm https://tools.invitrogen.com/content/sfs/gallery/low/g001619.jpg


Documents

Biosafety of Nanoparticles · Biosafety of Nanoparticles: Evaluation of Nanomaterials By a Suite of Cellular Assays Mellisa Theodore 1, Nathan Boggs , Bakhtiyor Rasulev PhD2, Jerzy