Cytotoxic and Immunologic Cytotoxic and Immunologic Effects of Silver NanoparticlesEffects of Silver Nanoparticles

Shaun CoteShaun Cote

What are Nanoparticles?What are Nanoparticles?

Any substance that has one dimension of Any substance that has one dimension of 1-100 nanometers1-100 nanometers

Commonly used nanoparticles include Commonly used nanoparticles include colloidal gold and titanium dioxide (TiOcolloidal gold and titanium dioxide (TiO22))

Nano ScaleNano Scale

Why is this important?Why is this important?

““The biokinetics of NSPs are different from The biokinetics of NSPs are different from larger particles. When inhaled, they are larger particles. When inhaled, they are efficiently efficiently deposited in all regions of the deposited in all regions of the respiratory tractrespiratory tract; they ; they evade specific defense evade specific defense mechanismsmechanisms; and they can ; and they can translocate out of translocate out of the respiratory tractthe respiratory tract via different pathways and via different pathways and mechanisms (endocytosis and transcytosis).”mechanisms (endocytosis and transcytosis).”

Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles.Environmental Health Perspectives, Oberdörster et al. 113(7), July 2005.

Downsides to Silver NanoparticlesDownsides to Silver Nanoparticles

Possible negative effects due to the Possible negative effects due to the chemistry of small moleculeschemistry of small molecules

How they are MadeHow they are Made

Metal salt Metal salt solutions are solutions are boiled and then boiled and then reduced via a reduced via a reducing agentreducing agent

Silver Nanoparticles in the MarketSilver Nanoparticles in the Market

SamsungSamsung Washing machine, refrigerator and air Washing machine, refrigerator and air

conditionersconditioners

NanohorizonsNanohorizons Smart Silver – silver nanoparticles intertwined Smart Silver – silver nanoparticles intertwined

with fabric to repel odor and microbeswith fabric to repel odor and microbes

Aigon Aigon SilverClene anti-microbial cleaning solutionSilverClene anti-microbial cleaning solution

Project GoalsProject Goals

To explore the effects of silver To explore the effects of silver nanoparticles by testing the:nanoparticles by testing the: Cytotoxicity on established cell lines:Cytotoxicity on established cell lines: Production of specific immune moleculesProduction of specific immune molecules

TestingTesting

1)1) MTT Assay – tests for metabolic activityMTT Assay – tests for metabolic activity

2)2) TNF-TNF-αα ELISA – Antibody test for TNF- ELISA – Antibody test for TNF-αα secretion from cellssecretion from cells

3)3) RT-PCR – Measure of RNA activity RT-PCR – Measure of RNA activity

Cell Lines TestedCell Lines Tested

RAW 264.7 – Mouse macrophageRAW 264.7 – Mouse macrophage Jurkat – Human T cellsJurkat – Human T cells WHTBF-6 – Human lung fibroblastWHTBF-6 – Human lung fibroblast

MTT AssayMTT Assay Cells are cultured, then treated with silver Cells are cultured, then treated with silver

nanoparticlesnanoparticles MTT is added, and blue crystals are MTT is added, and blue crystals are

formed if the cells are viableformed if the cells are viable Correlation between concentration of Correlation between concentration of

nanoparticles andnanoparticles andviability of the cellsviability of the cells

MTT Assay - ResultsMTT Assay - ResultsRAW 264.7 Cell Line

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Concentration

Per

cen

t V

iab

ility

Jurkat Cell Line

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Concentration

Per

cen

t V

iab

ility

WHTBF-6 Cell Line

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Concentration

Per

cen

t V

iab

ility

TNF-TNF-αα ELISA ELISA

Test based on antibody capture of TNF-Test based on antibody capture of TNF-αα, , a protein molecule essential in triggering a protein molecule essential in triggering an immune responsean immune response

Measured OD of sample wells compared Measured OD of sample wells compared to a standard curveto a standard curve

TNF-TNF-αα ELISA Results ELISA Results

0

50

100

150

200

250

300

350

400

C C-2.0 C-4.0 0.125 0.25 0.5 1.0 2.0 4.0 LPS

>60,000

Concentration Concentration of silver of silver nanoparticles nanoparticles versus versus relative relative concentration concentration of TNF-of TNF-αα

RT-PCRRT-PCR

A method of amplifying target RNA to A method of amplifying target RNA to determine how silver nanoparticles affect determine how silver nanoparticles affect gene expression of specific cytokinesgene expression of specific cytokines

Results of RT-PCRResults of RT-PCR

Only tested GAPDH, a housekeeping Only tested GAPDH, a housekeeping genegene

Further tests include the cytokines TNF-α, Further tests include the cytokines TNF-α, IL-6, and IL-1β IL-6, and IL-1β

Further StudiesFurther Studies

Continuing RT-PCR studies on other Continuing RT-PCR studies on other cytokinescytokines

AcknowledgementsAcknowledgements

Dr. Ah-Kau Ng and LabDr. Ah-Kau Ng and Lab Theresa KnightTheresa Knight Jennifer WalkerJennifer Walker SURF programSURF program