Content:

1. Overview of the latest application of spectroscopic methods for studying the interaction of nanoparticles with NA Lukáš Nejdl Datum konání: 7.3.2014

2. Content: • Spectroscopicmethods • New potentialdrug - platinumnanoparticles (PtNPs) induces DNA damage and inhibitionofTaq DNA polymerase • Raman spectroscopy • CharacterizationofPtNPs • Inhibition of Taq DNA polymerase (multiinstrumentalanalysis) • DNA damage (Komet essay) • of S. aureus and human fibroblasts (HFF) • Hematologicalanalysisoferythrocytes • The proposed mechanism of action PtNPs • Summary and conclusion

3. Spectroscopic methods: Neutron spin echo spectroscopy Mössbauer spectroscopy Neutron spin echo spectroscopy Photoacoustic spectroscopy Photoemission spectroscopy Photothermal spectroscopy Pump-probe spectroscopy Raman spectroscopy Saturated spectroscopy Scanning tunneling spectroscopy Spectrophotometry Time-resolved spectroscopy Thermal infrared spectroscopy Ultraviolet photoelectron spectroscopy Video spectroscopy Vibrational circular dichroism spectroscopy X-ray photoelectron spectroscopy Auger electron spectroscopy Acoustic resonance spectroscopy Cavity ring down spectroscopy Coherent anti-Stokes Raman spectroscopy Cold vapour atomic fluorescence spectroscopy Correlation spectroscopy Deep-level transient spectroscopy Photoacoustic spectroscopy Electron paramagnetic resonance spectroscopy Force spectroscopy Dual polarisation interferometry Fourier transform spectroscopy Hyperspectral imaging Inelastic electron tunneling spectroscopy Laser-Induced Breakdown Spectroscopy Mass spectroscopy Hadron spectroscopy

4. Raman spectroscopy - Raman spectroscopy is a spectroscopictechnique used to observe vibrational, rotational, and other low-frequency modes in a system.[ It relies on inelastic scattering, or Raman scattering, of monochromaticlight, usually from a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with molecular vibrations, phononsor other excitations in the system, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the vibrational modes in the system - je to mimořádně vhodná metoda pro zkoumání vztahu mezi strukturou a funkcí biomolekul

5. Raman spectroscopy Chandrasekhara Venkata Raman (*1888 and † 1970)

6. New potential drug - platinum nanoparticles (PtNPs) induces DNA damage and inhibition of Taq DNA polymerase • Characterization of PtNPs A B Kβ Kα C D a b c d e f gh a b d e f gh c

7. b) Inhibition of Taq DNA polymerase (multi instrumental analysis) PtNPs polymerase reaction CisPt polymerase reaction A B DNA Nanoplatin 0 Taq DNA Polymerase Cisplatin D C 0.040.42453420 420042000 0.040.20.4124.2 424204200 Ladder Ladder 500 bp 300 bp 100 Ladder 4200 8200 12400 1640020000 24000 28000 32000 36000 42000 Ladder 420 82012401640 2000 24002800 3200 18 19 500 bp 300 bp 100 Ladder 8200 8600 22500 9400 9800 10200 10600 11000 11400 12400 Ladder 420 460500540 580620660 700 500 bp 300 bp E F G 0 Melting point (°C) PtNPs (µg.mL-1) Pt-derivatives (µg.mL-1)

8. New potential drug - platinum nanoparticles (PtNPs) induces DNA damage and inhibition of Taq DNA polymerase 0 12.5 25 50 100 200 (µg.mL-1) c) DNA damage (Komet essay) 0 12.5 25 50 100 200 (µg.mL-1) 0 12.5 25 50 100 200 (µg.mL-1)

9. New potential drug - platinum nanoparticles (PtNPs) induces DNA damage and inhibition of Taq DNA polymerase d) The growth curves of S. aureus and human fibroblasts (HFF) B A e a a Relative change of growth rate (%) Relative change of growth rate (%) d e b b d c c Time (h) Time (h) C D % of survival % of survival Pt-derivatives (100 µg.mL-1 of Pt) Pt-derivatives (100 µg.mL-1 of Pt)

10. New potential drug - platinum nanoparticles (PtNPs) induces DNA damage and inhibition of Taq DNA polymerase e) Hematological analysis of erythrocytes

11. New potential drug - platinum nanoparticles (PtNPs) induces DNA damage and inhibition of Taq DNA polymerase f) The proposed mechanism of PtNPs

12. Summary and conclusion • In this work we proved that PtNPs primarily discards the activity of Taq DNA Polymerase and secondary damages DNA structure • We tend to believe that this effect together with the transition of PtNPs to Pt2+ causes mutagenicity and increased DNA damage of PtNPs in comparison to CisPt • The cytotoxic effect of PtNPs may be increased by encapsulation of PtNPs into the liposome • The results suggest p53 activation in PtNPs treated cells due to genotoxic stress, with subsequent activation of p21 leading to a proliferating cell nuclear antigen-mediated growth arrest in S phase and following apoptosis • It is conceivable that PtNPs with effective antitumor activity may provide an alternate treatment for cancer.

13. Acknowledgment Grant agency Financial support from CEITEC CZ.1.05/1.1.00/02.0068, NanoBioMetalNet and Liga proti rakovine LPR 2014 ishighlyacknowledged. Acknowledgment Prof. Ing. Rene Kizek, Ph.D. Doc. RNDr. Pavel Kopel,Ph.D Mgr. Dagmar Chudobová Mgr. Marie Konečná, Ph.D Dr. Ing. Branislav-RuttkayNedecky Ing. Kristýna Šmerková Bc. Simona Dostálová Ing. JiříKudr Mgr. Renata Kenšová, Ph.D. Ing. Soňa Krížkova, Ph.D. Lukáš Melichar Ing. Markéta Komínková

14. Thank you for your attention