F LUORESCENCE M ICROSCOPY : M EMBRANES AND P HARMACEUTICALS Nicole Antczak University of Connecticut CHEM 5395 March 3, 2001
O UTLINE Fluorescence Overview Fluorescence Microscope Confocal Micoscopy Fluorophores FRET Torchilin study
F LUORESCENCE – J ABLONSKI D IAGRAM
C ONFOCAL M ICROSCOPY
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
W ITH S OFTWARE …
F LUOPHORES Fluorescein/DAPI/Texas Red GFP and related proteins Quantum Dots
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
L IPID M IXING 4
T ORCHILIN S TUDY 5 Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate
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?
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
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
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
R ESULTS
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
T ESTING INTEGRITY OF TAT-L IPSOME C OMPLEX Rh-PE labeled liposomes with entrapped FTIC- dextran Colocalization of Rh and FTIC fluoresence
I NTRACELLULAR L OCALIZATION : A FTER 1 HOUR
2 HOURS
4 H OURS
9 HOURS
C ONCLUSIONS Can be used to follow targeting of liposomes and micelles Can be used for live cell imaging
C ITATIONS es/fluorescence/fluorhome.html es/fluorescence/fluorhome.html 3. es/confocal/index.html es/confocal/index.html 4. Lakowicz 5. Torchilin, V. P. Advanced Drug Delivery Reviews 57 (2005) 95– 109