1. Cellular and Molecular response to Nanoparticle Exposure By: Jewels Morgan

2. What is a nanoparticle? Small particles with a dimension of less than 100nm Small particles with a dimension of less than 100nm

3. Nanoparticles Uses Used in many different applications Used in many different applications –Biomedical –Optical –Electronics –Hygiene Products –Safety Products –Food Industry

4. Cross biological membrane Cross biological membrane Small surface area/volume ratio Small surface area/volume ratio Toxicity to human tissues and cell cultures Toxicity to human tissues and cell cultures Possible DNA interactions, damage, mutations Possible DNA interactions, damage, mutations Uptake by mitochondria and possible damage Uptake by mitochondria and possible damage Cell death Cell death What are possible dangers of Nanoparticle Exposure?

5. Previous Research Cultured two different cell lines Cultured two different cell lines –CHO (Chinese Hamster Ovary) –COS-7 (African Green Monkey Kidney Cells) Morphology of both cell lines Morphology of both cell lines Cytotoxicity of both cell lines Cytotoxicity of both cell lines Bead location Bead location Preliminary Apoptosis Preliminary Apoptosis

6. Particles Used Latex Beads, Blue dyed Latex Beads, Blue dyed Ultra fine nanoparticles (55nm) Ultra fine nanoparticles (55nm) Fine particles (800nm) Fine particles (800nm)

7. Fall 2007 Research Results Cytotoxicity Results Cytotoxicity Results –CHO 24 hrs –COS-7 48 hrs Both bead sizes showed cytotoxic affects to cells Both bead sizes showed cytotoxic affects to cells Morphology Morphology –Cultured cells morphology matched ATCC database Nanoparticle Location Nanoparticle Location –Determined localization of the nanoparticles to be in the cytoplasmic region to be in the cytoplasmic region

8. Pictures of Research Results CHO 24 hr exposure Bead Location Morphology

9. Current Research Spring 2008 Apoptosis Apoptosis –CHO cells –CASP1 (Caspase) Chromosomal Spread-Aberrations Chromosomal Spread-Aberrations –Halted in metaphase using colchicine Gene Expression of Pro-Inflammatory Cytokines-PCR RNA Gene Expression of Pro-Inflammatory Cytokines-PCR RNA –IL-6 –IL-18 Protein Concentration Protein Concentration

10. Apoptosis Programmed Cell death Programmed Cell death CHO Cells CHO Cells –Treated for 24 hrs with 1μL of beads –Isolated DNA –Verified purity and concentration Spectrophotometer Spectrophotometer λ260nm and λ280nm λ260nm and λ280nm

11. Treatment DNA Concentration DNA λ260nm/280nm Ratio Control 1.635μg/μL 1.89 Control 2.945 μg/μL 2.12 55nm beads 1.650 μg/μL 1.88 55nm beads 2.280 μg/μL 1.86 800nm beads 1.635 μg/μL 1.44 800nm beads 1.880 μg/μL 1.61 DNA Purity All values determined using a 1:100 dilution Ratio value 1.60 to 1.80=pure DNA

12. DNA Analysis following nanoparticle treatment Showed no fragmentation of DNA Showed no fragmentation of DNA No significant evidence of Apoptosis occurring No significant evidence of Apoptosis occurring Ladder 55nm CHO DNA

13. Pro-Inflammatory Cytokines Isolated RNA Isolated RNA –TRI Reagent Lyse cells to release RNA Lyse cells to release RNA –RNA Isolated from Controls, 55nm, & 800nm exposure cells –PCR samples with designed primers for: IL-6 IL-6 IL-18 IL-18 Caspase Caspase Beta Actin Beta Actin

14. IL-6 IL-6 –Released following oxidative stress to cell IL-18 IL-18 –Released following oxidative stress to cell CASPASE CASPASE –Elevated during Apoptosis Pro-Inflammatory Cytokines and Apoptotic gene

15. Primer Design NCBI Database NCBI Database –Chinese Hamster (Cricetulus griseus) IL-6 –Mouse (Mus musculus) Caspase -1 Unknown Sequence in CHO Unknown Sequence in CHO –Mouse (Mus musculus) IL-18 Unknown Sequence in CHO Unknown Sequence in CHO –Chinese Hamster (Cricetinae gen. sp) Beta Actin Endogenous gene expression Endogenous gene expression

16. Primer Design cont. Primers need to have: Primers need to have: –50% or greater gc content –Annealing temperature ~60.0ºC –Product size ~350-450bp Primers designed using Primer 3 software after importing sequence from NCBI database Primers designed using Primer 3 software after importing sequence from NCBI database Primer size 20bp Primer size 20bp

17. RNA Purity and Concentration Results Treatment RNA Concentration RNA λ260nm/280nm Ratio Control 1.572μg/μL 2.07 Control 2.136 μg/μL 1.48 55nm beads 1.656 μg/μL 2.82 55nm beads 2.144 μg/μL 2.00 800nm beads 1.202 μg/μL 1.80 800nm beads 1.164 μg/μL 1.78 All values determined using a 1:100 dilution Ration value of 1.8-2.0=Pure RNA

18. Gene Expression CHO IL-6 gene Ladder CHO CAS gene Ladder 1000bp 500bp Con 55nm 800nm 1000bp 500bp Con 55nm 800nm

19. Pro-Inflammatory Cytokine Response cont. Ladder Control 55nm 800nm IL-18 Beta Actin Ladder Control 55nm 800nm

20. Chromosomal Spreads

21. Chromosomal Spread- Aberrations Control CHO Chromosomes55nm Chromosome Spread 800nm Chromosome Spread

22. * Significantly different from control student’s t-test *

23. Protein Quantification Treatment Protein Concentration Control 1.750mg/mL Control 2.765mg/mL 55nm beads 1.840mg/mL 55nm beads 2.910mg/mL 800nm beads 1.905mg/mL 800nm beads 2.909mg/mL

24. Summary DNA Apoptosis DNA Apoptosis –No detection of Apoptotic bands –Increase in Caspase expression Cytokine Expression Cytokine Expression –IL-6 increased following treatment with 55nm beads compared to control –IL-18-No detection of gene expression Slight protein concentration in treated samples Slight protein concentration in treated samples Chromosome number and appearance altered following treatment Chromosome number and appearance altered following treatment

25. Special Thanks to…. Dr. Jacqueline Jordan-Research Mentor Dr. Jacqueline Jordan-Research Mentor Truyen Derek Pham-Supplies Donation Truyen Derek Pham-Supplies Donation –Emory University Sheryl Sands-Lab Technician Sheryl Sands-Lab Technician Dr. Michelle Furlong Dr. Michelle Furlong CSU Natural Sciences Faculty & Staff CSU Natural Sciences Faculty & Staff