A7906

Assays were performed as previously described (Hrycaj et al., 2018a (link)). Non-tissue culture treated polystyrene 96-well flat bottom microtiter plates (Denville, # T1097) were coated and incubated with bovine plasma fibronectin (Sigma-Aldrich, #F1141) at 20 μg/ml for 1 h at 37°C. Plates were then blocked with 100 μl/well of 1% BSA (Sigma-Aldrich, #A7906) in serum-free DMEM/F12 (Gibco, #11320033) for 30 min at 37°C. Fibroblasts were seeded at 10,000 cells per individual well. Plates were centrifuged (top side up) at 10 × g for 5 min to reduce the variability inherent in the settling of cells onto the plate surface and were incubated for 1 h at 37°C with 5% CO₂. Non-adherent cells were removed by centrifugation (top side down) at 48 × g for 5 min. Adherent cells remaining on the plate were fixed and stained with 1% formaldehyde (Fisher Sci., #BP228-100), 0.5% crystal violet (Sigma-Aldrich, #C6158), 20% MeOH followed by PBS washes. Individual wells were imaged on a Leica MZ125 dissecting microscope and manually quantified using ImageJ 2.0 cell counter function.

Release studies were carried out using different media, including PBS (pH 7.4, Gibco), 100% FBS (Sigma, F1051), 1% FBS in PBS pH 7.4, and 3.6% BSA (Sigma, A7906) in PBS (pH 7.4, Gibco). Release buffer volumes, total drug loadings, and sample dimensions are indicated in the figure captions. Sink conditions were maintained across all of the in vitro release studies through frequent buffer changes, ensuring that the released drug concentration was always at least five times lower than the solubility limit of the drug in the release buffer being used in order to avoid changes in release kinetics caused by drug levels approaching their saturation limit. Samples were incubated in release medium at 37 °C under constant agitation on an orbital shaker at 115 rpm (VWR). At designated timepoints, sampling was performed by complete removal of medium and replenishment with fresh buffer. Sampling timepoints included 24 h and then every 3–4 days thereafter. For PBS release conditions, samples were analyzed directly. For samples with protein (100% FBS, 1% FBS, 3.6% BSA), protein was precipitated by addition of acetonitrile. Samples were vortexed and centrifuged to separate extracted drug from precipitated protein. The supernatant was then analyzed for drug concentration by HPLC.