I've been trying to develop my technique as a graduate student and am attempting to partition the antifungal drug Nystatin into a liposome preparation consisting of 80% POPC/ 20% cholesterol. I've used literature to find a protocol for making the liposomes. Calculated volumes of POPC and Cholesterol in Chloroform were combined in a tube and dried under Nitrogen followed by overnight vacuum. I then used 50 mM Tris, 10 mM EDTA Buffer to dissolve the lipid film at 37 degrees Celsius for 3 minutes. This was followed by 3 rounds of 30 minutes at 37 degrees and 30 minutes at 4 degrees Celsius. Liposomes were then flushed with argon, wrapped with teflon tape and parafilm before use. My experiment consisted of preparing various concentrations of liposome using Tris buffer to dilute, followed by adding Nystatin to a final concentration of 10 uM. These solutions were incubated for one hour in a water bath at 37 degrees. A fluorometer with excitation slit width 1 nm, emission slit width 8 nm was used to take measurements of fluorescence. Excitation beam was 315 nm, emission was 415 nm with a quartz cuvette. All samples were prepared in parallel with references to which no drug was added. The literature suggests that there should be a significant enhancement of fluorescence that corresponds to partitioning of the drug in the membrane with respect to references, but I have not been able to accomplish this. Any suggestions for troubleshooting?
You don't seem to be doing anything in your procedure to reduce the size of the liposomes from multilamellar vesicles to unilamellar vesicles. As a result, the liposomes are very large and scatter a lot of light, making fluorescence measurements problematic. Try reducing the size of the liposomes by extrusion through~100 nm filters, or by sonication. Smaller liposomes produce a more transparent solution that is more suitable for fluorescence measurements.
Since you are adding nystatin to preformed liposomes, you can use liposomes without nystatin to subtract the baseline signal due to scattered light. Then it should be easier to discern whether there is more nystatin fluorescence with liposomes than without.
You might also try adding the nystatin into the lipid mixture from the start, and compare the fluorescence to that of liposomes without nystatin.
You should measure the whole emission spectrum to make sure any signal you see at 415 nm is really due to nystatin.
You may have to adjust the concentrations of nystatin and liposomes to get into a range where you don't have too much light scattering from the liposomes or too much inner filter effect from nystatin.
Ok, so I can try to sonicate a batch of liposomes that I have prepared. My PI published a paper using MLV, so it's been a little tricky to make the case that a different preparation may be necessary. I have been preparing two sets of samples for each experiment. I have a control where I don't add nystatin and subtract this from the value of the corresponding vial with nystatin. I have also done emission scans to see if there is any enhancement. There was one occasion where there was a nice enhancement in the 370nm-415nm range of emission wavelengths, but I have been unable to repeat the particular experiment successfully. When I run emission scans of the reference and sample for a given concentration of liposome, I scale the graphs and superimpose them. In the experiments I have run for the most part, the curves look almost identical, with no enhancement in the sample containing nystatin. I have a Branson water sonicator, are there any protocols that describe the duration of sonication? I haven't found much in the literature.
Put 1 ml of liposomes in a ~10-ml conical glass tube with screw cap or ground glass stopper. Flush with nitrogen and cover tightly. Use a clamp to position the tube so that the surface of the liposome suspension is at the same level as the surface of the water. Make sure the water level is adjusted so that you get plenty of agitation of the water's surface when the sonicator is on. Sonicate for 15-20 minutes. If it works, the suspension will go from opaque like cream to translucent like skim milk as the size of the liposomes decreases.
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