This study investigates the interaction of test drugs with caseum, a necrotic material from tuberculosis lesions. The methodology involves rapid equilibrium dialysis to quantify drug concentration and assess the unbound drug fraction.
Take a homogeneous suspension of caseum-the necrotic, lipid-rich material from tuberculosis lesions, which harbors drug-tolerant mycobacteria at its core.
Add the test drug to the homogenate and vortex to facilitate its interaction with the caseum matrix.
Place a rapid equilibrium dialysis insert-containing donor and receiver chambers separated by a semi-permeable membrane-into the base plate.
Load the drug-spiked homogenate into the donor chamber and add buffer to the receiver chamber.
Seal the plate and incubate with rotation.
During incubation, the free drug diffuses across the membrane, reaching equilibrium between both chambers.
Once equilibrium is reached, dilute the donor sample with buffer and the receiver sample with drug-free homogenate to normalize matrix conditions for drug quantification.
Finally, quantify the drug concentration and calculate the unbound drug fraction or fu.
The fu reflects the free drug available to diffuse into caseum and reach drug-tolerant mycobacteria.
Add 6.5 microliters of the prepared test compound to 643.5 microliters of the homogenate. To achieve the final concentration of five micromolar and vortex.
Next, place the red inserts into the base plate. Prepare the inserts for each test, each compound in triplicate.
Add 200 microliters of the drug-spiked matrix into the red donor chamber of each red insert. Then, add 350 microliters of PBS into each receiver chamber.
Place an adhesive seal on the plate and incubate it at 37 degrees Celsius on the thermal mixer at 200 rotations per minute for four hours. After the incubation, gently mix the contents of the donor and receiver chambers by pipetting up and down two to three times. Transfer 20 microliters of homogenate from each of the donor chambers to 20 microliters of clean PBS in a 1.5 milliliter tube.
Similarly, transfer 20 microliter aliquots of PBS samples from the receiver chambers to 20 microliters of clean homogenate.
Matrix matching eliminates the need for two separate color vision curves in homogenate and PBS to be made for quantitative analysis. Contents of the donor chamber may sediment over time. Gently mix the contents by pipetting before removing aliquots.
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