FLUORESCENCE MICROSCOPY MEMBRANES AND PHARMACEUTICALS Nicole Antczak University

FLUORESCENCE MICROSCOPY: MEMBRANES AND PHARMACEUTICALS Nicole Antczak University of Connecticut CHEM 5395 March 3, 2001

OUTLINE Fluorescence Overview Fluorescence Microscope Confocal Micoscopy Fluorophores FRET Torchilin study

FLUORESCENCE – JABLONSKI DIAGRAM http: //www. olympusmicro. com/primer/java/jablonski/jabintro/index. html

http: //www. olympusmicro. com/primer/techniques/fluorescence/anatomy/bx 51 fluorescence. html

http: //www. olympusmicro. com/primer/techniques/fluorescence/anatomy/ix 70 fluorescence. htm

CONFOCAL MICROSCOPY http: //www. olympusmicro. com/primer/techniques/confocalintro. html

CONFOCAL MICROSCOPY 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

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

WITH SOFTWARE… http: //www. olympusmicro. com/primer/techniques/confocalintro. html

http: //www. olympusmicro. com/moviegallery/confocal/rk 13 mkoh 2 b/index. html

FLUOPHORES Fluorescein/DAPI/Texas Red GFP and related proteins Quantum Dots

FLUORESCENCE RESONANCE ENERGY TRANSFER 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

LIPID MIXING 4

TORCHILIN STUDY 5 Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate

LIPOSOMES 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?

MICELLES 5 Colloidal dispersions of particle size within to 5 to 100 nm 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

ENHANCING MICELLE CELLULAR UPTAKE Cationic lipid formulations Lipofectin (LL) PEG-PE micelles carry negative charge Combination of both could improve uptake

DELIVERY OF VITAMIN K 3 MICELLES Vitamin K 3 (VK 3) inhibits growth of various cancer cell types May be involved in arylation of cellular thiols DBU used to accelerate formation of thioether of VK 3 analogs Does it have synergystic effects? Prepared micelles with both DBU and VK 3 and micelles with just VK 3

RESULTS

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

TESTING INTEGRITY OF TAT-LIPSOME COMPLEX Rh-PE labeled liposomes with entrapped FTICdextran Colocalization of Rh and FTIC fluoresence

INTRACELLULAR LOCALIZATION: AFTER 1 HOUR

2 HOURS

4 HOURS

9 HOURS

CONCLUSIONS Can be used to follow targeting of liposomes and micelles Can be used for live cell imaging

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