Victoria ZherdevaA.N.Bach Institute of Biochemistry of the Russian Academy of Science 28th Feb 1st March 2013 New Delhi International Seminar

Molecular biologyCell biologyHistologyPhysiologyMedicine InformationlevelTomographySesitivity to malecular events$$$$$$$$$-$$

CTnoUSnoNMR10-3 - 10-5 Fl10-9 - 10-12

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

Functional studies of proteins in living cellsConstruct target-GFP fusion proteinExamine at high resolution the behaviour of the protein in living cells

The technology of transgenic models obtainingTrancduced humanTumor cell line with gene of FPXenotransplantation of the fluorescent cell line to the Nude mouseFluorescent imaging techniquesTransfection (liposomal or lentiviral) of cancer cells with fluorescent repoter gene

Fluorescence imaging techniquesLaser spectrometer with optic fiber zondFluorescence diffuse tomographyiBox UVP

In vivo visualization of subcutaneous transduced models of lung adenocarcinoma 549-TagRFP on iBox (USA, UVP) on the 7-th, 15-th, 20-th day after tumor cells inoculation. Ex. filter 502-547 nm, em. filter 570-640 nm. Exposure time -1s7-th day15-th day20-th day

Monitoring of subcutaneous transduced models of lung adenocarcinoma 549-TagRFP () and 549-TRK23(B) with laser spectrometer SpectrClaster (Russia) on the 1-st, 7-th, 15-th, 20-th, 27-th, 32-d day after tumor cells inoculation. AB

mel Kor-Turbo-RFP cellsPhotosensitizer (PS)in vitroPreliminary screening of phototoxic action of PS on monolayers of fluorescent tumor cellsin vivo BalbC/Nu mice, female, 18-20 g, 8 weekCell inoculationTumor growth monitoringDiffuse fluorescent tomographiBoxexcitation 502-547 nm,emission 570-640 nm

Tumor fluorescenceFluorescence of Tiosenseexcitation 600-645 nm,emission > 700 nmexcitation 502-547 nm,emission 570-640 nm

Laser irradiation730 nm, 260 mWt/sm2, 20 min4 mg/kg of bodyweight

FRET eficciency FRET 51,1%* - Rusanov AL. et al. Lifetime imaging of FRET between red fluorescent proteins. J Biophotonics. 2010; 3:774-83

5

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KFP

Tag-RFP

,

1

TagRFPKFP

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6948.4737471008

6958.5524265097

6968.2720103266

6977.9628387554

6988.0193470183

6997.5252983257

7007.6007052134

1

KFP

Tag-RFP

,

2

3

549-agRFP549-RK23Lentivirus transfection

FLIMFLIM-FRETLife-time of donor, ns12

Intact cellsA549-TRK23A549-TRK23After apoptosis induction 800 H2O2 ,after 24 1.8-2.1 1.8-2.1 2.4-2.6

Small animal FLIM-FRET whole body imagingRed Fluorescent proteins (RFP)

Semiconducter core: Cd/Se, Cd/Te, and Ga/Nshell: Zn/S, Cd/Sebiomolecule:polymer, protein, lipidQuantum dots (QD) are nanometers size ( 1 10nm) semiconductor nanostructured materials with the tuneable size-dependent emission, high photoluminescence (PL) quantum yields, long PL lifetimes (1050ns) and narrow symmetric emission bands.Task 4: biodistribution of QD

Qd applicationMichalet 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.

QDs MPA em 611n or 630 n d ~ 8 11QY 10-20%QDs PolyT em 626 d ~ 15 16 QY 10-30%QDs PolyT-APS em 678 d ~ 36 QY 5-20%

Excititation 502-547 n, registration 570-640 , exposition 25 s.per os

The relative estimation: QDs were not detected (-), low amount of QDs (), well detectable amount of QDs (+).Fiber optical fluorescence spectroscopyFluorescence spectra of feces probes 24 h after per os administrationblack curve - feces control, red curve - feces after administration of QDs.

QDsStomach after 2 hIntestine after 2 hIntestine after 4 hIntestine after 6 hMPA+---PolyT++-PolyT-APS++++

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 Myllyl. 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.3885Publications:

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. 54598. 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.

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Imaging technology arise from the medical imaging from one side and micrMedical imagingis the technique and process used to createimagesof thehuman bodyfor clinical purposes or medical scienceFrom another hand it is a informative instruments in biology like microscopy.We used to recon them as routine methods. But in past 2 decades they gained new meaning with repoter molecules.A lot of probes ar used in MI: radionuclide, magnetic, fluorescentAlso they could be synthetic or semisynthetic or genetically encodedIn general I like to tell about different tasks which could be solved by methods of fluorescent imaging

**Imaging modalities can be broadly divided into primarily morphological/anatomical and primarily molecular imaging techniques. Primarily morphological/anatomical imaging technologies, such as computed tomography (CT), MRI (with contrast agents injected at millimolar blood concentrations) and ultrasound, are characterized by high spatial resolution. However, they also share thelimitation of not being able to detect diseases until tissue structural changes (for example, growth of a tumour) are large enough to be detected by the imaging modality.Primarily molecular imaging modalities, such as optical imaging, PET and SPECT (with radiotracers injected at nanomolar blood concentrations), offer the potential to detect molecular and cellular changes of diseases (for example, before the tumour is large enough to cause structural changes). However, these modalities suffer from a poor spatial resolution with currently availabletechnology. Combining the strengths of morphological/anatomicaland molecular imaging modalities (using multimodalityhardware and/or co-registration post-acquisition processing)allows the detection of pathophysiological changesin early disease phases at high structural resolution (forexample, PETCT and PETMRI technology).

Each modality has particular characteristics,advantages and limitations, which are highlighted here. Most imaging modalities are used clinically and can be translatedfrom animals to humans in the drug development process. The different imaging modalities are generally consideredcomplementary rather than competitive. See Supplementary information S1 (box) for a more detailed summary.CT, computed tomography; PET, positron-emission tomography; SPECT, single-photon-emission computed tomography.*On average, for ~10,000 compounds evaluatedin preclinical studies, about five compounds enter clinical trials and about one compound finally receives regulatoryapproval by the US Food and Drug Administration (FDA)3. The mean time from synthesis of a new compound to marketingapproval in the United States is 14.2 years137. Molecular imaging can be used at various stages in the drug developmentprocess, as illustrated here, which may help reduce attrition rates and allow the selection of the most promising drugcandidates early on in development.*Noday molecular imaging combained methods of biological and medical imaging from one side an**

GFP protein a was discovered in 1960-1970. There propertiers wereimproved on 1995/ AflGFPlike proteins were discovered later/The most important thing that is the whole structure of the protein is expressed like one gene/ So any molecule could be marked expressing in on e frame with target protein/**Subsequent subcutaneous inoculation of tumor cells to nude mice allows to monitor tumor growth in real time mode. Pseudocolor mapping helps to estimate the intensity distribution in tumor. The distribution of fluorescence intensity indicates the initial signs of the formation of new tumor nodes and metastasis. *For all tumor models good corrlelation between tumor growth and integral fluorescence are obtained. This indicates that transduction did not affect this morphological properties of cells.*Identification of characteristic protein spectra was performed with a laser spectrometer. The increase in fluorescence signal in both cases can be attributed to the growth of tumor depth. Thus, monitoring of fluorescent tumors can track the dynamics of growth of the tumor, its invasion. Genetically encoded sensor kaspazy3 will allow efficient screening of anticancer drugs in their sensitivity to kaspaze3. This approach can provide invaluable assistance at the stage of preclinical drug trials.* UVP. iBox (UVP, USA) , . ImageJ 1.42q (NIH, ), . * , , . mel Kor-TurboRFP ( 4 / ) 730 . * - . , , -3 , , , , . , . * 23 . KFP. . * . FLIM ( ) . . FRET , , . , . . , . , , . . . - , * TRK23. , , . , , , , . . , , . .** . , . . . gfhcfcf** - per os , , . , - , [V. Karabanovas, E. Zakarevicius, A. Sukackaite, G. Streckyte and R. Rotomskis, Photochem. Photobiol. Sci., 2008, 7, 725729]. . . , , . iBox -. 3- , 10 2 MPA, 4 PolyT PolyT-APS . 10 , 20 . , . , , . - (, , ) .

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