Fluorescence molecular imaging: the tasks and perspectives

1. Fluorescence molecular imaging: the tasks and perspectives Victoria Zherdeva A.N.Bach Institute of Biochemistry of the Russian Academy of Science 28th Feb – 1st March 2013 New Delhi International Seminar

2. DNA Proteins Cells Tissue Animal Human Genomics Proteomics Cytomics Phenomic Clinic Molecular biology Cell biology Histology Medicine Physiology Information level Sesitivity to malecular events Tomography $$ $$ $$$$ $-$$

3. A summary of modalities used for molecular imaging. Nature Reviews: Drug discovery. 7: 591-606; J. K. Willmann et al., Molecular imaging in drug development

4. к Nature Reviews: Drug discovery. 7: 591-606; J. K. Willmann et al., Molecular imaging in drug development

5. Fluorescence molecular imaging in vivo microscopy cellular level target reporter stop DNA mRNA Whole-body imaging fusion proteins

6. Functional studies of proteins in living cells • Construct target-GFP fusion protein • Examine at high resolution the behaviour of the protein in living cells

7. Number of publications on color proteins according to PubMed 2008 Nobel Prize in Chemistry "for the discovery and development of the green fluorescent protein, GFP“. Osamu Shimomura Marine Biological Laboratory Martin Chalfie Columbia University Roger Y. Tsien University of California, San Diego

8. The technology of transgenic models obtaining Trancduced human Tumor cell line with gene of FP Transfection (liposomal or lentiviral) of cancer cells with fluorescent repoter gene Xenotransplantation of the fluorescent cell line to the Nude mouse Fluorescent imaging techniques

9. Fluorescence imaging techniques Laser spectrometer with optic fiber zond Fluorescence diffuse tomography iBox UVP

10. Task 1: Tumor monitoring 7-th day 15-th day 20-th day In vivo visualization of subcutaneous transduced models of lung adenocarcinoma А549-TagRFP on iBox (USA, UVP) on the 7-th, 15-th, 20-thday after tumor cells inoculation. Ex. filter 502-547 nm, em. filter 570-640 nm. Exposuretime -1s

11. Correlation between tumor growth and integral fluorescence of tumor models (iBox, NIH Image) А549-TRK23 (A) and А549- TagRFP(Б)

12. A B Monitoring of subcutaneous transduced models of lung adenocarcinoma А549-TagRFP (А) and А549-TRK23(B) with laser spectrometerSpectrClaster (Russia) on the 1-st, 7-th, 15-th, 20-th, 27-th, 32-dday after tumor cells inoculation.

13. Task2. In vivo PDT (photodynamic therapy) agents screening in vitro in vivo Preliminary screening of phototoxic action of PS onmonolayers of fluorescent tumor cells Cell inoculation mel Kor-Turbo-RFP cells Photosensitizer (PS) Diffusefluorescent tomograph BalbC/Numice, female, 18-20 g, 8 week Tumor growth monitoring iBox excitation 502-547 nm, emission 570-640 nm

14. Co-localization of fluorescent tumor and photosensitizer (UVP iBox) Fluorescence of “Tiosense” Tumor fluorescence excitation 502-547 nm, emission 570-640 nm excitation 600-645 nm, emission > 700 nm

15. PDT after i.v. injection of liposomal “Tiosense” 4 mg/kg of bodyweight Laser irradiation 730 nm, 260 mWt/sm2, 20 min

16. Task 3: Molecular visualization of enzyme activities Caspase dependent apoptosis

17. FRET-sensor (genetically encoded fluorescent substrate) * - Rusanov AL. et al. Lifetime imaging of FRET between red fluorescent proteins. J Biophotonics. 2010; 3:774-83 FRET eficciencyFRET 51,1% TagRFP

18. PCMV IE KFP TagRFP GTGGSGGDEVDGTGGSGDPPVAT Lung adenocarcinomaА549transduced by pLVT –TagRFPandpLVT –TRK23 , 7 пассаж. Nikon Eclipse TE 2000-U, 20х, exp.1/5 s Lentivirus transfection А549-ТagRFP А549-ТRK23

19. FLIM FLIM-FRET 1 2 MicroTime 200, PicoQuant Life-time of donor, ns

20. FRET-FLIM 1.8-2.1 нс Intact cells A549-TRK23 A549-TRK23 After apoptosis induction 800 мкм H2O2 ,after 24 ч 1.8-2.1 нс 2.4-2.6нс

21. Small animal FLIM-FRET whole body imaging Red Fluorescent proteins (RFP)

22. Task 4: biodistribution of QD Quantum dots (QD) are nanometers size ( 1– 10nm) semiconductor nanostructured materials with the tuneable size-dependent emission, high photoluminescence (PL) quantum yields, long PL lifetimes (10–50ns) and narrow symmetric emission bands. Semiconducter core: Cd/Se, Cd/Te, and Ga/N shell: Zn/S, Cd/Se biomolecule: polymer, protein, lipid

23. Qd application Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S: Quantum Dots for Live Cells, in Vivo Imaging, and Diagnositics. Science 2005 307(5709):538-544.

24. QDs MPA • λem 611nм or 630 nм • d ~ 8 – 11нм • QY 10-20% QDs PolyT • λem 626 нм • d ~ 15 – 16 нм • QY 10-30% QDs PolyT-APS • λem 678 нм • d ~ 36 нм • QY 5-20%

25. In vivo monitoring of Qd fluorescence in digestion tract of mice per os Excititation 502-547 nм, registration 570-640 нм, exposition 25 s.

26. Fiber optical fluorescence spectroscopy The relative estimation: QDs were not detected (-), low amount of QDs (±), well detectable amount of QDs (+). Fluorescence spectra of feces probes 24 h after per os administration QDs MPA QDs PolyT QDs PolyT-APS black curve - feces control, red curve - feces after administration of QDs.

27. Publications: • 1. A.L. Rusanov, T.V. Ivashina, L.M. Vinokurov, I.I. Fiks, A.G. Orlova, I.V. Turchin, I.G. Meerovich, V.V. Zherdeva, and A.P. Savitsky. Lifetime imaging of FRET between red fluorescent proteins. J. Biophotonics, 2010, v. 3(12), p. 774-783. • 2. A.L. Rusanov, A.P. Savitsky. Fluorescence resonance energy transfer between fluorescent proteins as powerful toolkits for in vivo studies. Las. Phys. Lett., 2011, v. 8(2), p. 91-102. • 3. Rusanov A.L., Mironov V.A., Goryashenko A.S., Grigorenko B.L., Nemukhin A.V., Savitsky A.P. «Conformational partitioning in pH-induced fluorescence of the kindling fluorescent protein (KFP)» // J Phys Chem B. (2011);115(29):9195-201. • 4. Alexander L. Rusanov, Tatiana V. Ivashina, Leonid M. Vinokurov, Alexander S. Goryashenko, Victoria V. Zherdeva, Alexander P. Savitsky «FRET-sensor for imaging with lifetime resolution» // Laser Applications in Life Sciences, edited by Matti Kinnunen; Risto Myllylä. Proceedings of the SPIE, Volume 7376, pp. 737611-1-6 (2010). • 5. Alexander P. Savitsky, Alexander L. Rusanov, Victoria V. Zherdeva, Tatiana V. Gorodnicheva, Maria G. Khrenova and Alexander V. Nemukhin. FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins. Theranostics. (2012) v. 2, №2, pp.215-226. doi:10.7150/thno.3885

28. 6. Loginova Y.F., Kazachkina N.I., Zherdeva V.V., Rusanov A.L., Shirmanova M.V., Zagaynova E.V., Sergeeva E.A., Dezhurov S.V., Wakstein M.S., Savitsky A.P. Biodistribution of intact fluorescent CdSe/CdS/ZnS quantum dots coated by mercaptopropionic acid after intravenous injection into mice. – J. Biophotonics, 2012, vol. 11-12, pp. 848-859. 7. Loginova Y.F., Dezhurov S.V., Zherdeva V.V., Kazachkina N.I., Wakstein M.S., Savitsky A.P. Biodistribution and stability of CdSe core quantum dots in mouse digestive tract following per os administration: Advantages of double polymer/silica coated nanocrystals. – Biochem. Biophys. Res. Comm., 2012, vol. 419 (1), pp. 54–59 8. Salykina Y.F., Zherdeva V.V., Dezhurov S.V., Wakstein M.S., Shirmanova M.V., Zagaynova E.V., Martyanov A.A., Savitsky A.P. Biodistribution and clearance of quantum dots in small animals. – Proc. SPIE, 2011, vol. 7999, pp. 799908 – 799908-10.

29. Thank you