1. F LUORESCENCE M ICROSCOPY : M EMBRANES AND P HARMACEUTICALS Nicole Antczak University of Connecticut CHEM 5395 March 3, 2001

2. O UTLINE Fluorescence Overview Fluorescence Microscope Confocal Micoscopy Fluorophores FRET Torchilin study

3. F LUORESCENCE – J ABLONSKI D IAGRAM http://www.olympusmicro.com/primer/java/jablonski/jabintro/index.html

4. http://www.olympusmicro.com/primer/techniques/fluorescence/anatomy/bx51fluorescence.html

5. http://www.olympusmicro.com/primer/techniques/fluorescence/anatomy/ix70fluorescence.html

6. C ONFOCAL M ICROSCOPY http://www.olympusmicro.com/primer/techniques/confocal/confocalintro.html

7. C ONFOCAL M ICROSCOPY 1 Uses laser scanning Limited number of laser wavelengths Allows for scanning not only in x-y but in z direction produce thin (0.5 to 1.5 micrometer) optical sections through fluorescent specimens that have a thickness ranging up to 50 micrometers or more

8. http://www.olympusmicro.com/primer/techniques/confocal/confocalintro.html

9. W ITH S OFTWARE … http://www.olympusmicro.com/primer/techniques/confocal/confocalintro.html

10. http://www.olympusmicro.com/moviegallery/confocal/rk13mkoh2b/index.html

11. F LUOPHORES Fluorescein/DAPI/Texas Red GFP and related proteins Quantum Dots

12. F LUORESCENCE R ESONANCE E NERGY T RANSFER Occurs between donor molecule and acceptor molecule Can be used to measure distance between two sites on a macromolecule Also used to detect protein-protein interactions Membrane fusion and lipid exchange

13. L IPID M IXING 4

14. T ORCHILIN S TUDY 5 Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate

15. L IPOSOMES 5 Artificial phospholipid vesicles Loaded with water soluble drugs or water insoluble drugs Typically use antibodies for targeting Long circulated liposomes coated with PEG Why not combine the two?

16. M ICELLES 5 Colloidal dispersions of particle size within to 5 to 100nm Able to increase solubility and bioavailability of poorly soluble pharmaceuticals Several key micelle properties for drug incorporation Size, CMC, load capacity of hydrophobic core Small size advantageous PEG/antibodies

18. E NHANCING M ICELLE C ELLULAR U PTAKE Cationic lipid formulations Lipofectin (LL) PEG-PE micelles carry negative charge Combination of both could improve uptake

19. D ELIVERY OF V ITAMIN K3 M ICELLES Vitamin K3 (VK3) inhibits growth of various cancer cell types May be involved in arylation of cellular thiols DBU used to accelerate formation of thioether of VK3 analogs Does it have synergystic effects? Prepared micelles with both DBU and VK3 and micelles with just VK3

20. R ESULTS

21. TAT-L IPOSOMES Trans-activating transcriptional activator protein (TAT) Attached TAT-peptide to liposomes via PEG/PE molecule Several hundred TAT-peptides with single 200nm liposome

23. T ESTING INTEGRITY OF TAT-L IPSOME C OMPLEX Rh-PE labeled liposomes with entrapped FTIC- dextran Colocalization of Rh and FTIC fluoresence

24. I NTRACELLULAR L OCALIZATION : A FTER 1 HOUR

25. 2 HOURS

26. 4 H OURS

27. 9 HOURS

28. C ONCLUSIONS Can be used to follow targeting of liposomes and micelles Can be used for live cell imaging

29. C ITATIONS 1. http://www.olympusmicro.com/index.html http://www.olympusmicro.com/index.html 2. http://www.olympusmicro.com/primer/techniqu es/fluorescence/fluorhome.html http://www.olympusmicro.com/primer/techniqu es/fluorescence/fluorhome.html 3. http://www.olympusmicro.com/primer/techniqu es/confocal/index.html http://www.olympusmicro.com/primer/techniqu es/confocal/index.html 4. Lakowicz 5. Torchilin, V. P. Advanced Drug Delivery Reviews 57 (2005) 95– 109