Cellular and Molecular Cellular and Molecular response to response to Nanoparticle ExposureNanoparticle Exposure

By: Jewels MorganBy: Jewels Morgan

What is a What is a nanoparticle?nanoparticle? Small particles with a dimension Small particles with a dimension

of less than 100nmof less than 100nm

Nanoparticles UsesNanoparticles Uses

Used in many different Used in many different applications applications – BiomedicalBiomedical– OpticalOptical– ElectronicsElectronics– Hygiene ProductsHygiene Products– Safety ProductsSafety Products– Food IndustryFood Industry

Cross biological membraneCross biological membrane Small surface area/volume ratioSmall surface area/volume ratio Toxicity to human tissues and cell Toxicity to human tissues and cell

culturescultures Possible DNA interactions, damage, Possible DNA interactions, damage,

mutationsmutations Uptake by mitochondria and possible Uptake by mitochondria and possible

damagedamage Cell deathCell death

What are possible What are possible dangers of Nanoparticle dangers of Nanoparticle Exposure?Exposure?

Previous ResearchPrevious Research

Cultured two different cell linesCultured two different cell lines– CHO (Chinese Hamster Ovary)CHO (Chinese Hamster Ovary)– COS-7 (African Green Monkey COS-7 (African Green Monkey

Kidney Cells)Kidney Cells) Morphology of both cell linesMorphology of both cell lines Cytotoxicity of both cell lines Cytotoxicity of both cell lines Bead location Bead location Preliminary ApoptosisPreliminary Apoptosis

Particles UsedParticles Used

Latex Beads, Blue dyedLatex Beads, Blue dyed Ultra fine nanoparticles (55nm)Ultra fine nanoparticles (55nm) Fine particles (800nm)Fine particles (800nm)

Fall 2007 Research Fall 2007 Research ResultsResults Cytotoxicity ResultsCytotoxicity Results

– CHO 24 hrsCHO 24 hrs– COS-7 48 hrsCOS-7 48 hrs

Both bead sizes showed cytotoxic affects to cellsBoth bead sizes showed cytotoxic affects to cells MorphologyMorphology

– Cultured cells morphology matched ATCC Cultured cells morphology matched ATCC databasedatabase

Nanoparticle LocationNanoparticle Location– Determined localization of the Determined localization of the

nanoparticles nanoparticles to be in the cytoplasmic regionto be in the cytoplasmic region

Pictures of Research Pictures of Research ResultsResults

CHO 24 hr exposure

Bead Location

Morphology

Current Research Spring Current Research Spring 20082008

ApoptosisApoptosis– CHO cellsCHO cells– CASP1 (Caspase)CASP1 (Caspase)

Chromosomal Spread-AberrationsChromosomal Spread-Aberrations– Halted in metaphase using colchicineHalted in metaphase using colchicine

Gene Expression of Pro-Inflammatory Gene Expression of Pro-Inflammatory Cytokines-PCR RNACytokines-PCR RNA– IL-6IL-6– IL-18IL-18

Protein ConcentrationProtein Concentration

ApoptosisApoptosis

Programmed Cell deathProgrammed Cell death CHO Cells CHO Cells

– Treated for 24 hrs with 1Treated for 24 hrs with 1μμL of beadsL of beads– Isolated DNAIsolated DNA– Verified purity and concentrationVerified purity and concentration

SpectrophotometerSpectrophotometer λλ260nm and 260nm and λλ280nm 280nm

TreatmentTreatment DNA DNA ConcentrationConcentration

DNA DNA λλ260nm/280nm 260nm/280nm

RatioRatio

Control 1Control 1 .635.635μμg/g/μμLL 1.891.89

Control 2Control 2 .945 .945 μμg/g/μμLL 2.122.12

55nm beads 155nm beads 1 .650 .650 μμg/g/μμLL 1.881.88

55nm beads 255nm beads 2 .280 .280 μμg/g/μμLL 1.861.86

800nm beads 1800nm beads 1 .635 .635 μμg/g/μμLL 1.441.44

800nm beads 1800nm beads 1 .880 .880 μμg/g/μμLL 1.611.61

DNA PurityDNA Purity

All values determined using a 1:100 dilutionRatio value 1.60 to 1.80=pure DNA

DNA Analysis following DNA Analysis following nanoparticle treatmentnanoparticle treatment

Showed no Showed no fragmentation of fragmentation of DNADNA

No significant No significant evidence of evidence of Apoptosis Apoptosis occurringoccurring

Ladder 55nm

CHO DNA

Pro-Inflammatory Pro-Inflammatory CytokinesCytokines Isolated RNAIsolated RNA

– TRI ReagentTRI Reagent Lyse cells to release RNALyse cells to release RNA

– RNA Isolated from Controls, 55nm, & RNA Isolated from Controls, 55nm, & 800nm exposure cells800nm exposure cells

– PCR samples with designed primers for:PCR samples with designed primers for: IL-6IL-6 IL-18IL-18 CaspaseCaspase Beta Actin Beta Actin

IL-6IL-6– Released following oxidative stress Released following oxidative stress

to cell to cell IL-18IL-18

– Released following oxidative stress Released following oxidative stress to cellto cell

CASPASECASPASE– Elevated during Apoptosis Elevated during Apoptosis

Pro-Inflammatory Pro-Inflammatory Cytokines and Cytokines and Apoptotic geneApoptotic gene

Primer DesignPrimer Design

NCBI Database NCBI Database – Chinese Hamster Chinese Hamster (Cricetulus griseus) (Cricetulus griseus)

IL-6IL-6– Mouse (Mouse (Mus musculus) Mus musculus) Caspase -1Caspase -1

Unknown Sequence in CHOUnknown Sequence in CHO

– Mouse (Mouse (Mus musculus) Mus musculus) IL-18IL-18 Unknown Sequence in CHOUnknown Sequence in CHO

– Chinese Hamster (Chinese Hamster (Cricetinae gen. sp) Cricetinae gen. sp) Beta ActinBeta Actin Endogenous gene expressionEndogenous gene expression

Primer Design cont.Primer Design cont.

Primers need to have:Primers need to have:– 50% or greater gc content50% or greater gc content– Annealing temperature ~60.0Annealing temperature ~60.0ºCºC– Product size ~350-450bpProduct size ~350-450bp

Primers designed using Primer 3 Primers designed using Primer 3 software after importing software after importing sequence from NCBI databasesequence from NCBI database

Primer size 20bpPrimer size 20bp

RNA Purity and RNA Purity and Concentration ResultsConcentration Results

TreatmentTreatment RNA RNA ConcentrationConcentration

RNA RNA λλ260nm/280nm 260nm/280nm

RatioRatio

Control 1Control 1 .572.572μμg/g/μμLL 2.072.07

Control 2Control 2 .136 .136 μμg/g/μμLL 1.481.48

55nm beads 155nm beads 1 .656 .656 μμg/g/μμLL 2.822.82

55nm beads 255nm beads 2 .144 .144 μμg/g/μμLL 2.002.00

800nm beads 1800nm beads 1 .202 .202 μμg/g/μμLL 1.801.80

800nm beads 1800nm beads 1 .164 .164 μμg/g/μμLL 1.781.78

All values determined using a 1:100 dilutionRation value of 1.8-2.0=Pure RNA

Gene ExpressionGene Expression

CHO IL-6 gene

Ladder

Ladder

CHO CAS gene

Ladder

1000bp

500bp

Con 55nm 800nm

1000bp

500bp

Con 55nm 800nm

Pro-Inflammatory Pro-Inflammatory Cytokine Response Cytokine Response cont.cont.

Ladder

Control 55nm 800nm

IL-18 Beta Actin

Ladder Control 55nm 800nm

Chromosomal SpreadsChromosomal Spreads

Chromosomal Spread-Chromosomal Spread-AberrationsAberrations

Control CHO Chromosomes 55nm Chromosome Spread 800nm Chromosome Spread

* Significantly different from control student’s t-test

Chromosome Spread Analysis

0

2

4

6

8

10

12

14

16

Control 55 nm 800 nm

Treatments

Nu

mb

er

of

ch

rom

oso

mes *

Protein QuantificationProtein Quantification

TreatmentTreatment Protein ConcentrationProtein Concentration

Control 1Control 1 .750mg/mL.750mg/mL

Control 2Control 2 .765mg/mL.765mg/mL

55nm beads 155nm beads 1 .840mg/mL.840mg/mL

55nm beads 255nm beads 2 .910mg/mL.910mg/mL

800nm beads 1800nm beads 1 .905mg/mL.905mg/mL

800nm beads 2800nm beads 2 .909mg/mL.909mg/mL

SummarySummary

DNA ApoptosisDNA Apoptosis– No detection of Apoptotic bandsNo detection of Apoptotic bands– Increase in Caspase expressionIncrease in Caspase expression

Cytokine ExpressionCytokine Expression– IL-6 increased following treatment with 55nm IL-6 increased following treatment with 55nm

beads compared to controlbeads compared to control– IL-18-No detection of gene expressionIL-18-No detection of gene expression

Slight protein concentration in treated Slight protein concentration in treated samplessamples

Chromosome number and appearance Chromosome number and appearance altered following treatmentaltered following treatment

Special Thanks to….Special Thanks to…. Dr. Jacqueline Jordan-Research MentorDr. Jacqueline Jordan-Research Mentor Truyen Derek Pham-Supplies DonationTruyen Derek Pham-Supplies Donation

– Emory UniversityEmory University Sheryl Sands-Lab TechnicianSheryl Sands-Lab Technician Dr. Michelle FurlongDr. Michelle Furlong CSU Natural Sciences Faculty & StaffCSU Natural Sciences Faculty & Staff