Dear members,
Can you please help me out, how to visualize dye encapsulated liposomes. As my dye is weekly fluorescent nature in lipid vesicle will it be useful to take fluorescence or con focal microscopy pictures?? Is there any other techniques available to know whether my dye was encapsulated in liposomes or not? How to confirm it?
I would like to encapsulate my weekly fluorescent dye in interior of liposome and I have to confirm the dye encapsulation with any of the know technique then would like to see leakage experiments. Please suggest how to confirm this type of dye encapsulation by using know techniques (available instruments in my lab: UV-Vis , fluorescence spectrometer , fluorescence microscopy and DLS instrument). Is there any standard protocols for this? or please suggest some relative references.
Regards
Prathap
Hi Prathap, I suggest centrifuging the liposomes with moderate speed, e.g. ~10,000c g for 5-10 min, then resuspend the pellet in water or appropriate solvent, then measure the dye fluorescence in a spectrofluorimeter (confocal microscopy would be extremely time consuming for quantification). Then you could follow leakage comparing to this starting value. Best: Karoly
Hi Partap,
You can do one more thing. If you just want to know if your dye has been encapsulated into a liposome, you can compare the UV-Vis spectra of each of the individual items alone (e.g. the dye, material for synthesizing liposomes, the prepared liposome). Usually, the lambda max will be a bit different.
Once you do that, then the next step will be to study the study the release kinetics of the dye from the liposome. This study will also confirm that the dye has been encapsulated with the encapsulating material.
Please see the attached paper and links for further explanation.
http://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1016&context=gradschool_theses
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