Characterizing the transport properties of therapeutics and their carriers is critical for ensuring effective biological responses. These methods aid in designing optimally charged drug carriers for targeting negatively charged tissues. The main advantage of these techniques is their ability to better predict in vivo therapeutic efficacies through a series of in vitro experiments that characterize solute transport through tissue.
Cartilage diseases, such as osteoarthritis, remain untreated due to the inability of drugs to penetrate the dense avascular cartilage matrix, requiring drug carriers to prologue therapeutic efficacy. Begin by using a delicate task wipe to gently remove excess PBS from the surfaces of three millimeter diameter, one millimeter thick cartilage explants. Use a balance to quickly record the wet weight of each explant and immediately place the explants into a PBS bath to prevent dehydration.
Next, add 300 microliters of freshly prepared, 30 micromolar fluorescently labeled cationic peptide carrier solution per well to the inner wells of a 96-well plate and use a spatula to add one explant to each well of solution. Fill each of the surrounding wells with 300 microliters of PBS and cover the plate with a lid. Seal the edges of the plate with flexible film to minimize evaporation and place the plate on a shaker in a 37 degree Celsius incubator for 24 hours at 50 revolutions per minute, with a 15 millimeter orbit.
At the end of the incubation, transfer the equilibrium bath from each well into individual polypropylene tubes and make serial dilutions from the stock 30 micromolar cationic peptide carrier solution to generate a standard curve. Then, transfer 200 microliters of each solution and standard into individual wells of a black 96-well plate and obtain fluorescence readings of each sample and standard based on the excitation and emission wavelengths of the fluorescent label. To determine the depth of cationic peptide carrier penetration within a cartilage explant, use a scalpel to cut six millimeter diameter, one millimeter thick cartilage explants in half and hydrate the resulting half-disc pieces with proteus inhibitor supplemented PBS.
Apply epoxy to the center of a well of a custom-designed one-dimensional transport chamber and secure one half-disk explant within the well with the superficial side of the explant facing the upstream side of the chamber. Remove any excess glue from the well to prevent contact with the diffusion surface area of the cartilage and add 80 microliters of protease inhibitor supplemented PBS to both sides of the explant. Pipette the liquid up and down on one side of the explant to check for leakage to the other side.
If no leakage is present, replace the protease inhibitor supplemented PBS from the upstream side with 80 microliters of 30 micromolar fluorescence labeled cationic peptide carrier solution and carefully place the transport chamber into a cell culture dish. Cover the base of the dish with PBS to avoid cationic peptide carrier solution evaporation. Taking care that there is no direct contact between the solutions from the upstream and downstream chambers.
Place the covered dish on a shaker to limit particle sedimentation for four or 24 hours at room temperature and 50 revolutions per minute, with a 15 millimeter orbit. At the end of the incubation, remove the explants from the chamber and cut approximately 100 micron thick slices from the center of each explant. Place each slice of explant between a glass slide and a cover slip and hydrate the slices with fresh protease inhibitor supplemented PBS.
Fix the slide onto the stage of a confocal microscope and obtain a Z-stack of fluorescent images through the full thickness of the slice at 10X magnification. Open the image file and image J, click Image, and select Stacks and Z Project from the dropdown menu. Then, input the slice numbers from one to the final slice and under Projection Type, select average intensity and click Okay.
To assess the non-equilibrium cationic peptide carrier cartilage diffusion rate, assemble each half of the transport chamber, to include one large rubber gasket, one polymethylmethacrylate insert, and one small rubber gasket. Measure the thickness of a cartilage explant, then place the explant in the wells of the plastic insert with the superficial surface facing the upstream chamber and sandwich the two halves together to complete the assembly. Use a wrench to tightly screw the halves together before filling the upstream chamber with two milliliters of protease inhibitor supplemented PBS.
Check the downstream chamber for leakage from the upstream chamber. If no leakage is detected, fill the downstream chamber with two milliliters of protease inhibitor supplemented PBS. Add a single mini stir bar to both the up and downstream chambers and place the chamber on a stir plate with the chamber aligned such that the laser from the spectrophotometer is focused towards the center of the downstream chamber.
With the signal receiver portion of the spectrophotometer behind the downstream chamber, collect stable, realtime downstream fluorescence emission readings for at least five minutes. After obtaining a stable reading, add a pre-calculated volume of fluorescently labeled cationic peptide carrier stock solution to the upstream chamber to a final bath concentration of three micromolar, and monitor the downstream fluorescent signal while allowing the solute transport to reach a steady increase in slope. Once a steady state has been reached, transfer 20 microliters of solution from the upstream chamber to the downstream chamber for a spike test and collect the real-time downstream fluorescence readings.
Too high a positive charge will limit solute penetration to the superficial zone as the carrier binds too strongly to the negatively charged aggrecan groups of the cartilage matrix. Conversely, carriers that are able to take advantage of weak and reversible charge interactions penetrate through the deep zone of tissue. An optimally charged drug carrier, however, would not only penetrate the deep zones of tissue, but would also demonstrate a high intra-tissue uptake, which can be determined using the fluorescence measurements of the equilibrium bath and tissue wet weight, as shown.
Non-equilibrium diffusion transport experiments result in a data generated curve with a gradually increasing slope. The initial part of the curve represents solute diffusion through the cartilage as solute matrix binding interactions occur. Once solutes reach the downstream chamber, the slope of the curve increases as the fluorescence readings increase over time.
This second part of the curve then reaches a steady slope, representing steady state diffusion. The X-intercept of a tangential line drawn at the steady state portion of the curve indicates the time it takes to reach steady state diffusion or tau lag. Following solution transfer from the upstream to the downstream chamber, a spike in fluorescence is observed, at which point the stabilized fluorescence intensity can be used to correlate the fluorescence to the concentration.
The representative effective diffusivity and steady state diffusion values can then be calculated, as indicated. Note that the steady state diffusivity is two orders of magnitude higher than the effective diffusivity as a result of all of the charge-based binding sites being occupied. Thus, at this point, diffusion is based on size and not charge, resulting in similar steady state diffusion values among CPCs of same size.
Maintain explant hydration and minimize solution evaporation throughout the experiment to prevent changes in the cartilage morphology and the solution concentration, respectively, and to ensure accurate and reproducible data acquisition. Following the design of an optimally charged drug carrier, various conjugation techniques can be utilized to modify drugs for enhanced tissue targeting and to facilitate evaluation of their biological efficacy.
